Method for preventing and treating liver fibrosis

ABSTRACT

The present invention relates to a method for preventing and treating hepatic fibrosis, comprising administering an effective amount of plasminogen to a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a national stage filing under 35 U.S.C. 371 ofInternational Patent Application Serial No. PCT/CN2017/089056, filedJun. 19, 2017, which claims priority to International Patent ApplicationSerial No. PCT/CN2016/110174, filed Dec. 15, 2016. The entire contentsof these applications are incorporated herein by reference in theirentirety.

TECHNICAL FIELD

The present invention relates to a method for preventing and treatinghepatic fibrosis, comprising administering an effective amount ofplasminogen to a subject.

BACKGROUND ART

Fibrosis is a pathological change characterized by the activation andproliferation of fibroblasts, and increased fibrous connective tissuesand decreased parenchymal cells in tissues and organs. Continuousprogress can lead to structural destruction and loss of function oftissues and organs. Fibrosis of vital organs seriously affects thequality of life of patients and even endangers life. Tissue fibrosis isthe leading cause of disability and death in many diseases worldwide.According to the relevant statistics of the United States, about 45% ofthe deaths due to various diseases in this country can be attributed tothe diseases of tissue fibroplasia.

Fibrotic diseases comprise diseases affecting multiple systems, such assystemic sclerosis, multifocal fibrosis, scleroderma, and nephrogenicmultisystem fibrosis, and further comprise organ- and tissue-specificdiseases, such as skin, cardiac, pulmonary, hepatic, renal fibrosis,etc. Different fibrosis diseases have different etiologies, such astissue and organ injury, infection, immune response and chronicinflammation; however, their common characteristics are excessivedeposition of extracellular matrix (ECM) in tissues as well as organ andtissue remodeling^([1-3]).

Cardiac fibrosis occurs in the development and progression of a varietyof heart diseases, such as viral myocarditis, myocardial infarction, andhypertensive heart disease. Excessive cardiac fibrosis leads to impairedcardiac function and is the main cause of heart failure in manydiseases, wherein inhibition and reversal of myocardial fibrosis havebecome important links in the treatment of cardiovascular diseases.

Hepatic fibrosis refers to a pathological process of abnormalhyperplasia of connective tissue in the liver caused by variouspathogenic factors, and excessive deposition of diffuse extracellularmatrix in the liver. Hepatic fibrosis can be caused by a variety offactors, such as viral infection, inflammatory response, oxidativestress, and alcoholism. The pathological features of hepatic fibrosisare that there are large amounts of fibrous tissue hyperplasia anddeposition in the portal area and hepatic lobule, but no interlobularseptum has been formed. In hepatic cirrhosis, pseudolobules are formed,septa appear in the central venous area and the portal area, and thenormal structure of the liver is destroyed. Further progression ofhepatic fibrosis is called hepatic cirrhosis. Viral hepatitis is themain chronic liver disease in China. Hepatic tissue fibrosis in chronicviral hepatitis is associated with inflammation, necrosis, viralreplication and the like in the liver, and is reversible at an earlystage. Therefore, therapies such as antiviral therapy, body immunefunction adjustment, and protecting hepatocytes are combined withanti-fibrosis, which is an active measure to prevent hepatic fibrosis.

Pulmonary fibrosis diseases comprise idiopathic pulmonary fibrosis,sarcoidosis, allergic pneumonia, pneumoconiosis, drug-induced andradiation-induced fibrosis, and a broad spectrum of diseases withvarying etiologies such as fibrogenic alveolitis associated withcollagen vascular disease. The main pathological features comprise lungtissue mesenchymal cell proliferation, extracellular matrixproliferation and deposition, and remodeling of lung parenchyma. Atpresent, anti-inflammation, anti-oxidation, anti-fibroblastproliferation, collagen deposition, lung transplantation and othermeasures are mainly used to treat pulmonary fibrosis.

Renal fibrosis is a pathological process in which extracellular matrixand inappropriate connective tissue accumulate in the kidney, leading torenal structural changes and impaired functions. It is also a commonpathway for almost all renal diseases to progress to end-stage renalfailure. The process of renal fibrosis involves inflammatory response,apoptosis of innate cells and immune cells, imbalance of a variety ofregulatory factors of fibrosis, and the like; therefore, renal fibrosiscan be resisted through anti-inflammation, anti-apoptosis, treatmentagainst fibrosis factors and other ways.

Chronic lesions of tissues and organs are generally accompanied byfibrosis, for example, chronic inflammation and chronic lesions of thelung are accompanied by pulmonary fibrosis. Likewise, for hepaticfibrosis, for instance, hepatitis B, hepatitis C, alcoholic liver, fattyliver, schistosomiasis and the like are accompanied by early-stagehepatic fibrosis. Since the compensatory function of the liver is verystrong, fibrotic lesions have been latent in a variety of chronic liverdiseases and are generally discovered when they develop into hepaticcirrhosis. In fact, hepatic cirrhosis is a serious stage of hepaticfibrosis. Furthermore, for instance, chronic nephritis, glomerulitis,tubulitis and the like are all accompanied by renal fibrosis; andcardiovascular, cerebrovascular, and lower extremity vascular sclerosis,narrowing, or obstruction are all accompanied by vascular fibrosis.

Skin fibrosis forms scar tissues. Scar tissues are fibrous connectivetissues in the aging stage formed by the remodeling and maturation ofgranulation tissues. In the case of trauma, fibroblasts divide andproliferate, migrate to the damaged site, produce extracellular matrix,form scar tissue, and repair trauma.

The formation of scars is a process of progressive fibrosis ofgranulation tissues. As such, there are more and more reticular fibersand collagen fibers, and the reticular fibers become collagenized andthe collagen fibers become thicker; meanwhile, fewer and fewerfibroblasts are left, and a small number of the remaining ones aretransformed into fibrocytes; the interstitial fluid is graduallyabsorbed, and neutrophils, macrophages, lymphocytes and plasma cellsdisappear successively; and capillaries are closed, degenerated, anddisappear, leaving few arterioles and venules. In this way, thegranulation tissue is transformed into a scar tissue composed mainly ofcollagen fibers with few blood vessels, which is white and tough underthe naked eyes.

Scar tenacity and inelasticity, together with scar contraction can causeorgan deformation and dysfunction. Therefore, scarring around the jointsand vital organs often causes joint spasm or restricted movement, forinstance, lumen stenosis may be caused in the lumen organs such as thedigestive tract and the urinary tract, and movement disorder may becaused near the joints. Cicatricial adhesions between organs or betweenorgans and body cavity walls often affect their function to varyingdegrees. If extensive fibrosis and hyaline degeneration occur afterextensive injury in the organ, organ sclerosis will occur.

Systemic sclerosis (SSc), also known as scleroderma, is a systemicautoimmune disease characterized by localized or diffuse skin thickeningand fibrosis. The lesions are characterized by fibrous hyperplasia ofskin and onion skin changes of blood vessels, which eventually lead toskin sclerosis and vascular ischemia. The disease is clinicallycharacterized by localized or diffuse skin thickening and fibrosis. Inaddition to skin involvement, it can also affect the internal organs(heart, lungs, digestive tract and other organs).

Atherosclerosis generally leads to ischemic injuries of tissues andorgans, which in turn causes fibrotic lesions in tissues and organs.Atherosclerosis is a chronic, progressive arterial disease in which thefat deposited in the arteries partially or completely blocks blood flow.Atherosclerosis occurs when the otherwise smooth and solid arterialintima becomes roughened and thickened and is blocked by fat, fibrin,calcium, and cellular debris. Atherosclerosis is the chronicinflammatory hyperplasia of arterial intima, which leads to the stenosisor occlusion of large- and medium-sized arterial lumens, and causesischemic injuries, fibrosis and even necrosis of corresponding organsand tissues.

Atherosclerosis is closely related to diabetes mellitus, which ismanifested by early onset, severe degree and poor prognosis ofatherosclerosis in diabetic patients, and atherosclerosis is the maincause of death in diabetic patients. Clinically, it has been found thatthe pathological changes of coronary arteries in diabetic patients aresubstantially characterized by more affected vessels, severe coronaryartery stenosis, and more diffuse and severe lesions, and that themechanism is mostly attributed to abnormal glucose metabolism causingatherosclerosis. With further in-depth research, more results indicatethat diabetes mellitus-induced atherosclerosis is not caused by a singlefactor, but through a variety of pathways and more complex mechanisms toinduce and promote the development and progression of atherosclerosis,such as polarization of macrophages, macrophage migration inhibitoryfactor pathway, advanced glycation end products pathway, scavengerreceptor upregulation, insulin resistance, ubiquitin proteasome systemactivation, and platelet-derived growth factor activationpathway.^([4 ])

At present, a large number of people are suffering from fibrosisdiseases caused by various reasons, patients often have multiple organsand tissues affected, such diseases still lack effective therapies, andthe social and economic burdens are relatively heavy. The studies of thepresent invention found that plasminogen can ameliorate tissue and organfibrosis, and improve tissue and organ functions, thus opening up a newfield for preventing and treating tissue and organ fibrosis and itsrelated conditions.

SUMMARY OF THE INVENTION

The present invention comprises the following items:

1. A method for preventing or treating hepatic collagen deposition orfibrosis caused by a hepatic tissue injury in a subject, comprisingadministering an effective amount of plasminogen to the subject.

2. The method of item 1, wherein the injury comprises an ischemicreperfusion injury, a drug-induced injury, an immune injury, achemical-induced injury, an inflammatory injury, an excessivedrinking-induced injury, and a cancer-induced injury.

3. The method of item 2, wherein the injury comprises a fatdeposition-induced injury.

4. A method for preventing or treating hepatic collagen deposition orfibrosis in a subject, comprising administering an effective amount ofplasminogen to the subject.

5. A method for preventing or treating hepatic collagen deposition orfibrosis caused or complicated by diabetes mellitus in a subject,comprising administering an effective amount of plasminogen to thesubject.

6. A method for preventing or treating hepatic collagen deposition orfibrosis caused or complicated by atherosclerosis in a subject,comprising administering an effective amount of plasminogen to thesubject.

7. A method for preventing or treating hepatic collagen deposition orfibrosis caused or complicated by hyperlipemia in a subject, comprisingadministering an effective amount of plasminogen to the subject.

8. The method of item 7, wherein the hyperlipemia comprises one or moreselected from: an elevated blood triglyceride level, an elevated totalblood cholesterol level, increased blood low-density lipoprotein, andincreased blood very low-density lipoprotein.

9. A method for preventing or treating hepatic collagen deposition orfibrosis caused by a drug-induced hepatic injury in a subject,comprising administering an effective amount of plasminogen to thesubject.

10. The method of item 9, wherein the drug is a hepatotoxic drug.

11. The method of item 10, wherein the drug comprises a chemotherapeuticdrug, an antibiotic drug, a hypolipidemic drug, an antihypertensivedrug, and a hypoglycemic drug.

12. A method for preventing or treating alcoholic hepatic collagendeposition or fibrosis or non-alcoholic hepatic collagen deposition orfibrosis in a subject, comprising administering an effective amount ofplasminogen to the subject.

13. A method for preventing or treating hepatic collagen deposition orfibrosis caused by chronic hepatic disease in a subject, comprisingadministering an effective amount of plasminogen to the subject.

14. A method for preventing or treating hepatic collagen deposition orfibrosis caused by viral hepatitis in a subject, comprisingadministering an effective amount of plasminogen to the subject.

15. A method for preventing or treating a hepatic fibrosis-relatedcondition in a subject, comprising administering an effective amount ofplasminogen to the subject.

16. The method of item 15, wherein the hepatic fibrosis-relatedcondition comprises a condition resulting from decreased hepaticfunction, hepatic dysfunction or loss of hepatic function due to hepaticfibrosis.

17. The method of any one of items 1 to 16, wherein the plasminogen isadministered in combination with one or more other drugs or therapeuticmeans.

18. The method of item 17, wherein the other drugs comprise: ahypolipidemic drug, an anti-platelet drug, an antihypertensive drug, avasodilator, a hypoglycemic drug, an anticoagulant drug, a thrombolyticdrug, a hepatoprotective drug, an anti-fibrosis drug, an anti-arrhythmiadrug, a cardiotonic drug, a diuretic drug, an anti-tumor drug, aradiotherapeutic or chemotherapeutic drug, an inflammatory regulatorydrug, an immunomodulatory drug, an antiviral drug, and an antibiotic.

19. The method of any one of items 1 to 18, wherein the plasminogen hasat least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identitywith SEQ ID No. 2, 6, 8, 10 or 12, and still has the plasminogenactivity.

20. The method of any one of items 1 to 19, wherein the plasminogen is aprotein that has 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35,1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2 or 1 amino acid added,deleted and/or substituted in SEQ ID No. 2, 6, 8, 10 or 12, and stillhas the plasminogen activity.

21. The method of any one of items 1 to 20, wherein the plasminogen is aprotein that comprises a plasminogen active fragment and still has theplasminogen activity.

22. The method of any one of items 1 to 21, wherein the plasminogen isselected from Glu-plasminogen, Lys-plasminogen, mini-plasminogen,micro-plasminogen, delta-plasminogen or their variants that retain theplasminogen activity.

23. The method of any one of items 1 to 22, wherein the plasminogen is anatural or synthetic human plasminogen, or a variant or fragment thereofthat still retains the plasminogen activity.

24. The method of any one of items 1 to 22, wherein the plasminogen isan ortholog of human plasminogen from a primate or a rodent, or avariant or fragment thereof that still retains the plasminogen activity.

25. The method of any one of items 1 to 24, wherein the amino acids ofthe plasminogen are as shown in SEQ ID No. 2, 6, 8, 10 or 12.

26. The method of any one of items 1 to 25, wherein the plasminogen is anatural human plasminogen.

27. The method of any one of items 1 to 26, wherein the subject is ahuman.

28. The method of any one of items 1 to 27, wherein the subject has alack or deficiency of plasminogen.

29. The method of any one of items 1 to 28, wherein the lack ordeficiency is congenital, secondary and/or local.

30. A plasminogen for use in the method of any one of items 1 to 29.

31. A pharmaceutical composition, comprising a pharmaceuticallyacceptable carrier and the plasminogen for use in the method of any oneof items 1 to 29.

32. A preventive or therapeutic kit comprising: (i) the plasminogen foruse in the method of any one of items 1 to 29, and (ii) a means fordelivering the plasminogen to the subject.

33. The kit of item 32, wherein the means is a syringe or a vial.

34. The kit of item 32 or 33, further comprising a label or aninstruction for use indicating the administration of the plasminogen tothe subject to implement the method of any one of items 1 to 29.

35. An article of manufacture, comprising:

a container comprising a label; and

(i) the plasminogen for use in the method of any one of items 1 to 29 ora pharmaceutical composition comprising the plasminogen, wherein thelabel indicates the administration of the plasminogen or the compositionto the subject to implement the method of any one of items 1 to 29.

36. The kit of any one of items 32 to 34 or the article of manufactureof item 35, further comprising one or more additional means orcontainers containing other drugs.

37. The kit or the article of manufacture of item 36, wherein the otherdrugs are selected from the group of a hypolipidemic drug, ananti-platelet drug, an antihypertensive drug, a vasodilator, ahypoglycemic drug, an anticoagulant drug, a thrombolytic drug, ahepatoprotective drug, an anti-fibrosis drug, an anti-arrhythmia drug, acardiotonic drug, a diuretic drug, an anti-tumor drug, aradiotherapeutic or chemotherapeutic drug, an inflammatory regulatorydrug, an immunomodulatory drug, an antiviral drug, and an antibiotic.

In one aspect, the present invention relates to a method for preventingand/or treating collagen deposition or fibrosis of a tissue and an organand its related conditions in a subject, comprising administering aneffective amount of plasminogen to the subject, wherein the subject issusceptible to tissue and organ fibrosis, has a tendency of tissue andorgan fibrosis, or suffers from other diseases accompanied by tissue andorgan fibrosis. The present invention further relates to the use ofplasminogen for preventing and/or treating collagen deposition orfibrosis of a tissue and an organ and its related conditions in asubject. The present invention further relates to the use of plasminogenin the preparation of a medicament for preventing and/or treatingcollagen deposition or fibrosis of a tissue and an organ and its relatedconditions in a subject. In addition, the present invention furtherrelates to the plasminogen for preventing and/or treating collagendeposition or fibrosis of a tissue and an organ and its relatedconditions in a subject. In some embodiments, the collagen deposition orfibrosis of a tissue and an organ comprises skin fibrosis, vascularfibrosis, cardiac fibrosis, pulmonary fibrosis, hepatic fibrosis, andrenal fibrosis. In some other embodiments, the collagen deposition orfibrosis of a tissue and an organ comprises collagen deposition orfibrosis of a tissue and an organ elicited or accompanied by injuriescaused by infection, inflammation, hypersensitivity, tumors, tissueischemia, tissue and organ congestion, chemicals, radiation orenvironmental pollution. Specifically, the collagen deposition orfibrosis of a tissue and an organ comprises collagen deposition orfibrosis of a tissue and an organ caused by a tissue and organ lesiondue to a bacterial, viral or parasitic infection, wherein the collagendeposition or fibrosis of a tissue and an organ comprises pulmonaryfibrosis caused by Mycobacterium tuberculosis infection, hepaticfibrosis caused by a hepatitis B virus, hepatitis C virus or hepatitis Evirus infection, and hepatic fibrosis caused by schistosomiasisinfection. In some embodiments, the collagen deposition or fibrosis of atissue and an organ results from an aseptic inflammation or anautoimmune response. Specifically, the collagen deposition or fibrosisof a tissue and an organ is renal fibrosis caused by chronicglomerulonephritis, pyelonephritis, nephrotic syndrome, renalinsufficiency, and uremia. In some other embodiments, the collagendeposition or fibrosis of a tissue and an organ results from a tissueand organ injury caused by cancer. Specifically, the collagen depositionor fibrosis of a tissue and an organ is pulmonary fibrosis caused bylung cancer, hepatic fibrosis caused by liver cancer, or renal fibrosiscaused by kidney cancer. In some other embodiments, the collagendeposition or fibrosis of a tissue and an organ results from a chronicischemic tissue injury. Specifically, the collagen deposition orfibrosis of a tissue and an organ is cardiac ischemic fibrosis caused bycoronary atherosclerosis and coronary heart disease, and/or renalfibrosis caused by a chronic ischemic renal injury. In some otherembodiments, the collagen deposition or fibrosis of a tissue and anorgan results from tissue and organ congestion caused by acardiovascular disease. Specifically, the collagen deposition orfibrosis of a tissue and an organ is hepatic congestion or pulmonarycongestion. In some embodiments, the collagen deposition or fibrosis ofa tissue and an organ results from a drug. Specifically, the collagendeposition or fibrosis of a tissue and an organ is drug-induced hepaticfibrosis or renal fibrosis. In some embodiments, the collagen depositionor fibrosis of a tissue and an organ is pulmonary fibrosis caused byinhaled chemicals or environmental pollutants. In the above-mentionedembodiments, the collagen deposition or fibrosis of a tissue and anorgan results from a systemic immune disease such as systemic lupuserythematosus, systemic sclerosis, and ankylosing spondylitis. In someembodiments, the tissue and organ fibrosis is idiopathic pulmonaryfibrosis.

In the above-mentioned embodiments, the tissue and organfibrosis-related condition comprises a condition resulting from functionweakening, dysfunction or loss of function of a tissue or an organ dueto a fibrotic lesion. Specifically, the tissue and organfibrosis-related condition comprises atherosclerosis, coronary heartdisease, angina pectoris, myocardial infarction, arrhythmia, cerebralischemia, cerebral infarction, renal insufficiency, uremia, hepaticdysfunction, hepatic cirrhosis, hepatic coma, dyspnea, emphysema,pulmonary heart disease, pulmonary fibrosis, and ankylosing spondylitis.

In the above-mentioned embodiments, the plasminogen is administered incombination with one or more other drugs or therapies. Specifically, theplasminogen is administered in combination with one or more drugsselected from: a hypolipidemic drug, an anti-platelet drug, anantihypertensive drug, a vasodilator, a hypoglycemic drug, ananticoagulant drug, a thrombolytic drug, a hepatoprotective drug, ananti-fibrosis drug, an anti-arrhythmia drug, a cardiotonic drug, adiuretic drug, an anti-tumor drug, a radiotherapeutic orchemotherapeutic drug, an inflammatory regulatory drug, animmunomodulatory drug, an antiviral drug, and an antibiotic.

In the above-mentioned embodiments, the plasminogen has at least 75%,80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ IDNo. 2, 6, 8, 10 or 12, and still has the activity of plasminogen.

In the above-mentioned embodiments, the amino acids of the plasminogenare as shown in SEQ ID No. 2, 6, 8, 10 or 12. In some embodiments, theplasminogen is a protein that has 1-100, 1-90, 1-80, 1-70, 1-60, 1-50,1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2 or 1amino acid added, deleted and/or substituted in SEQ ID No. 2, 6, 8, 10or 12, and still has the activity of plasminogen.

In the above-mentioned embodiments, the plasminogen is a protein thatcomprises a plasminogen active fragment and still has the activity ofplasminogen. Specifically, the plasminogen is selected fromGlu-plasminogen, Lys-plasminogen, mini-plasminogen, micro-plasminogen,delta-plasminogen or their variants that retain the plasminogenactivity.

In the above-mentioned embodiments, the plasminogen is a natural orsynthetic human plasminogen, or a variant or fragment thereof that stillretains the plasminogen activity. In some embodiments, the plasminogenis an ortholog of human plasminogen from a primate or a rodent, or avariant or fragment thereof that still retains the plasminogen activity.For example, the plasminogen is an ortholog of plasminogen from primatesor rodents, for example, an ortholog of plasminogen from gorillas,rhesus monkeys, murine, cows, horses and dogs. Most preferably, theamino acid sequence of the plasminogen of the present invention is asshown in SEQ ID No. 2, 6, 8, 10 or 12.

In the above-mentioned embodiments, the subject is a human. In someembodiments, the subject has a lack or deficiency of plasminogen.Specifically, the lack or deficiency is congenital, secondary and/orlocal.

In one embodiment, the plasminogen is administered by systemic ortopical route, preferably by the following routes: topical, intravenous,intramuscular, subcutaneous, inhalation, intraspinal, local injection,intraarticular injection or rectal route. In one embodiment, the topicaladministration is performed by direct administration to osteoporoticareas, for example through a means such as a dressing and a catheter.

In one embodiment, the plasminogen is administered in combination with asuitable polypeptide carrier or stabilizer. In one embodiment, theplasminogen is administered at a dosage of 0.0001-2000 mg/kg, 0.001-800mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg/kg or 10-100mg/kg (by per kg of body weight) or 0.0001-2000 mg/cm², 0.001-800mg/cm², 0.01-600 mg/cm², 0.1-400 mg/cm², 1-200 mg/cm², 1-100 mg/cm² or10-100 mg/cm² (by per square centimeter of body surface area) daily,preferably the dosage is repeated at least once, preferably the dosageis administered at least daily. In the case of local administration, theabove dosages may also be further adjusted depending on thecircumstances. In one aspect, the present invention relates to apharmaceutical composition, comprising a pharmaceutically acceptablecarrier and the plasminogen for use in the method of the presentinvention.

In another aspect, the present invention relates to a preventive ortherapeutic kit comprising: (i) the plasminogen for use in the method ofthe present invention, and (ii) a means for delivering the plasminogento the subject, in particular, the means is a syringe or a vial. In someembodiments, the kit further comprises a label or an instruction for useindicating the administration of the plasminogen to the subject toimplement the methods of the present invention.

In another aspect, the present invention further relates to an articleof manufacture comprising: a container comprising a label; and (i) theplasminogen for use in the methods of the present invention or apharmaceutical composition comprising the plasminogen, wherein the labelindicates the administration of the plasminogen or the composition tothe subject to implement the methods of the present invention.

In the above-mentioned embodiments, the kit or the article ofmanufacture further comprises one or more additional means or containerscontaining other drugs. In some embodiments, the other drugs areselected from the group of: a hypolipidemic drug, an anti-platelet drug,an antihypertensive drug, a vasodilator, a hypoglycemic drug, ananticoagulant drug, a thrombolytic drug, a hepatoprotective drug, ananti-fibrosis drug, an anti-arrhythmia drug, a cardiotonic drug, adiuretic drug, an anti-tumor drug, a radiotherapeutic orchemotherapeutic drug, an inflammatory regulatory drug, animmunomodulatory drug, an antiviral drug, and an antibiotic.

DEFINITION

“Fibrosis” is a lesion characterized by the activation and proliferationof fibroblasts, and increased fibrous connective tissue and decreasedparenchymal cells in tissues and organs, and structural destruction andloss of function of tissues and organs, after the tissues and organssuch as lung, liver, kidney, blood vessel, peritoneum, pancreas and skinare continuously injured due to various causes such as inflammation,infection, immune response, ischemia, chemicals and radiation. The termcan be used interchangeably with “fibrotic lesion”. The term “fibroticlesion” encompasses fibrotic lesions in tissues and organs, such ascardiac fibrosis, pulmonary fibrosis, hepatic fibrosis, renal fibrosis,vascular fibrosis and skin fibrosis, which are caused by variousfactors, and further comprises the fibrotic lesions in tissues andorgans, such as cardiac fibrosis, pulmonary fibrosis, hepatic fibrosis,renal fibrosis, vascular fibrosis and skin fibrosis, which areassociated with the development and progression of various diseases.

After the development of fibrotic lesions in tissues and organs, theirnormal structures change, and the corresponding functions are weakenedor lost, and thus the resulting related conditions are called “tissueand organ fibrosis-related conditions”.

“Cardiac fibrosis” refers to the fibrotic lesion occurring in thedevelopment and progression of the cardiac tissue injury caused oraccompanied by various factors (such as inflammation, infection, immuneresponse, ischemia, chemicals, and radiation), or of heart diseasescaused by various factors. Cardiac fibrosis lesions lead to impairedcardiac function, and thus the resulting related conditions are called“cardiac fibrosis-related conditions”, including but not limited to thesymptoms and conditions of organ and tissue ischemia caused by impairedcardiac function, such as coronary heart disease, angina pectoris,myocardial infarction, arrhythmia, cerebral ischemia, dyspnea, and renalinsufficiency.

“Hepatic fibrosis” refers to pathological changes (lesions) caused oraccompanied by various factors (such as inflammation, infection (such asviral infection), immune response, ischemia, chemicals, radiation,oxidative stress, and alcoholism), comprising the abnormal hyperplasiaof connective tissue in the liver, excessive deposition of diffuseextracellular matrix in the liver, and destruction of the normalstructure of liver. Further progression of hepatic fibrosis is hepaticcirrhosis, and is also encompassed within the scope of the term “hepaticfibrosis” of the present invention. Hepatic fibrosis lesions lead toimpaired hepatic function, and thus the resulting related conditions arecalled “hepatic fibrosis-related conditions”.

“Pulmonary fibrosis” refers to a pathological process caused by lungtissue mesenchymal cell proliferation, extracellular matrixproliferation and deposition, and lung parenchymal remodeling which arecaused or accompanied by various factors (such as inflammation,infection, immune response, ischemia, chemicals, and radiation).Pulmonary fibrosis lesions lead to impaired pulmonary function, and thusthe resulting related conditions are called “pulmonary fibrosis-relatedconditions”.

“Renal fibrosis” refers to a pathological process in which abnormalaccumulation of connective tissues in the kidney caused or accompaniedby various factors (such as inflammation, infection, immune response,ischemia, chemicals, and radiation) occurs, leading to renal structuralchanges and impaired functions. The renal fibrosis lesion is a commonpathway by which almost all renal diseases progress to the later stages.

Renal fibrosis lesions lead to impaired renal function, and thus theresulting related conditions are called “renal fibrosis-relatedconditions”, for instance, renal insufficiency, renal failure, uremia,etc.

Chronic lesions of tissues and organs are generally accompanied byfibrosis, for example, chronic inflammation and chronic lesions of thelung are accompanied by pulmonary fibrosis. Likewise, for hepaticfibrosis, for example, hepatitis B, hepatitis C, alcoholic liver, fattyliver, schistosomiasis and the like are accompanied by early-stagehepatic fibrosis. Furthermore, for instance, chronic nephritis,glomerulitis, tubulitis and the like are all accompanied by renalfibrosis; and cardiovascular, cerebrovascular, and lower extremityvascular sclerosis, narrowing, or obstruction are all accompanied byvascular fibrosis. In the present invention, the term “fibrosis” or“fibrotic lesion” encompasses fibrotic lesions generally accompanied bychronic lesions of various tissues and organs of the body.

“Systemic sclerosis”, also known as “scleroderma”, is a systemicautoimmune disease characterized by localized or diffuse skin thickeningand fibrosis. The lesions are characterized by fibrous hyperplasia ofskin and onion skin changes of blood vessels, which eventually lead toskin sclerosis and vascular ischemia. The disease is clinicallycharacterized by localized or diffuse skin thickening and fibrosis. Inaddition to skin involvement, it can also affect the internal organs(heart, lungs, digestive tract and other organs).

“Atherosclerosis” is a chronic, progressive arterial disease in whichthe fat deposited in the arteries partially or completely blocks bloodflow. Atherosclerosis is a progressive process. When the concentrationof lipids in the blood is greatly increased, fatty streaks form alongthe arterial wall. These streaks can lead to deposits of fat andcholesterol, which attach to the otherwise smooth arterial intima andthus form nodules. Underneath these nodules, fibrotic scar tissuedevelops, leading to calcium deposition. The calcium deposits graduallydevelop into a chalky hard film (referred to as atherosclerotic plaque)that cannot be removed. When an artery connected to a tissue or an organin the body is blocked, the ischemic injury to the tissue or organcaused by the blocked artery in the tissue or organ may lead to afibrotic lesion of the tissue or organ, such as fibrosis of the heart,lung, liver, kidney, blood vessel, peritoneum, pancreas and skin.

Diabetes mellitus generally occurs with the development ofatherosclerosis, and the mechanism is mostly attributed toatherosclerosis caused by abnormal glucose metabolism. With furtherin-depth research, more results indicate that diabetes mellitus-inducedatherosclerosis is not caused by a single factor, but through a varietyof ways and a more complex mechanism to induce and promote thedevelopment and progression of atherosclerosis^([4]). Diabetes mellitusand its concomitant atherosclerosis may lead to tissue and organinjuries and fibrosis, such as fibrosis of the heart, lung, liver,kidney, blood vessel, peritoneum, pancreas, skin, and other tissues andorgans.

DETAILED DESCRIPTION OF EMBODIMENTS

Plasmin is a key component of the plasminogen activation system (PAsystem). It is a broad-spectrum protease that can hydrolyze severalcomponents of the extracellular matrix (ECM), including fibrin, gelatin,fibronectin, laminin, and proteoglycan^([5]). In addition, plasmin canactivate some pro-matrix metalloproteinases (pro-MMPs) to form activematrix metalloproteinases (MMPs). Therefore, plasmin is considered to bean important upstream regulator of extracellular proteolysis^([6,7]).Plasmin is formed by the proteolysis of plasminogen by two physiologicalPAs: tissue plasminogen activator (tPA) or urokinase-type plasminogenactivator (uPA). Due to the relatively high level of plasminogen inplasma and other body fluids, it is traditionally believed that theregulation of the PA system is primarily achieved through the levels ofPA synthesis and activity. The synthesis of PA system components isstrictly regulated by different factors, such as hormones, growthfactors and cytokines. In addition, there are also specificphysiological inhibitors of plasmin and PAs. The main inhibitor ofplasmin is α2-antiplasmin. The activity of PAs is simultaneouslyinhibited by the plasminogen activator inhibitor-1 (PAI-1) of uPA andtPA and regulated by the plasminogen activator inhibitor-2 (PAI-2) thatprimarily inhibits uPA. There are uPA-specific cell surface receptors(uPARs) that have direct hydrolytic activity on certain cellsurfaces^([8,9]).

Plasminogen is a single-stranded glycoprotein composed of 791 aminoacids and has a molecular weight of about 92 kDa^([10,11]) Plasminogenis mainly synthesized in the liver and is abundantly present in theextracellular fluid. The content of plasminogen in plasma is about 2 μM.Therefore, plasminogen is a huge potential source of proteolyticactivity in tissues and body fluids^([12,13]). Plasminogen exists in twomolecular forms: glutamic acid-plasminogen (Glu-plasminogen) andlysine-plasminogen (Lys-plasminogen). The naturally secreted anduncleaved forms of plasminogen have an amino-terminal (N-terminal)glutamic acid and are therefore referred to as glutamicacid-plasminogen. However, in the presence of plasmin, glutamicacid-plasminogen is hydrolyzed to lysine-plasminogen at Lys76-Lys77.Compared with glutamic acid-plasminogen, lysine-plasminogen has a higheraffinity for fibrin and can be activated by PAs at a higher rate. TheArg560-Va1561 peptide bond between these two forms of plasminogen can becleaved by uPA or tPA, resulting in the formation of plasmin as adisulfide-linked double-strand protease^([14]). The amino-terminalportion of plasminogen contains five homotrimeric rings, i.e., theso-called kringles, and the carboxy-terminal portion contains a proteasedomain. Some kringles contain lysine-binding sites that mediate thespecific interaction of plasminogen with fibrin and its inhibitor α2-AP.A newly discovered plasminogen is a 38 kDa fragment, comprising kringles1-4, is a potent inhibitor of angiogenesis. This fragment is named asangiostatin and can be produced by proteolysis of plasminogen by severalproteases.

The main substrate of plasmin is fibrin, and the dissolution of fibrinis the key to prevent pathological thrombosis^([15]). Plasmin also hassubstrate specificity for several components of ECM, including laminin,fibronectin, proteoglycan and gelatin, indicating that plasmin alsoplays an important role in ECM remodeling^([11,16,17]). Indirectly,plasmin can also degrade other components of ECM by converting certainprotease precursors into active proteases, including MMP-1, MMP-2, MMP-3and MMP-9. Therefore, it has been proposed that plasmin may be animportant upstream regulator of extracellular proteolysis^([18]). Inaddition, plasmin has the ability to activate certain potential forms ofgrowth factors^([19-21]). In vitro, plasmin can also hydrolyzecomponents of the complement system and release chemotactic complementfragments.

“Plasmin” is a very important enzyme that exists in the blood and canhydrolyze fibrin clots into fibrin degradation products and D-dimers.

“Plasminogen” is the zymogenic form of plasmin, and based on thesequence in the swiss prot and calculated from the amino acid sequence(SEQ ID No. 4) of the natural human plasminogen containing a signalpeptide, is a glycoprotein composed of 810 amino acids, which has amolecular weight of about 90 kD and is synthesized mainly in the liverand capable of circulating in the blood; and the cDNA sequence encodingthis amino acid sequence is as shown in SEQ ID No. 3. Full-lengthplasminogen contains seven domains: a C-terminal serine protease domain,an N-terminal Pan Apple (PAp) domain and five Kringle domains (Kringles1-5). Referring to the sequence in the swiss prot, the signal peptidecomprises residues Metl-Gly19, PAp comprises residues Glu20-Va198,Kringle 1 comprises residues Cys103-Cys181, Kringle 2 comprises residuesGlu184-Cys262, Kringle 3 comprises residues Cys275-Cys352, Kringle 4comprises residues Cys377-Cys454, and Kringle 5 comprises residuesCys481-Cys560. According to the NCBI data, the serine protease domaincomprises residues Va1581-Arg804.

Glu-plasminogen is a natural full-length plasminogen and is composed of791 amino acids (without a signal peptide of 19 amino acids); the cDNAsequence encoding this sequence is as shown in SEQ ID No. 1; and theamino acid sequence is as shown in SEQ ID No. 2. In vivo,Lys-plasminogen, which is formed by hydrolysis of amino acids atpositions 76-77 of Glu-plasminogen, is also present, as shown in SEQ IDNo.6; and the cDNA sequence encoding this amino acid sequence is asshown in SEQ ID No.5. δ-plasminogen is a fragment of full-lengthplasminogen that lacks the structure of Kringle 2-Kringle 5 and containsonly Kringle 1 and the serine protease domain^([22,23].) The amino acidsequence (SEQ ID No. 8) of δ-plasminogen has been reported in theliterature^([23]), and the cDNA sequence encoding this amino acidsequence is as shown in SEQ ID No. 7. Mini-plasminogen is composed ofKringle 5 and the serine protease domain, and has been reported in theliterature to comprise residues Va1443-Asn791 (with the Glu residue ofthe Glu-plasminogen sequence that does not contain a signal peptide asthe starting amino acid)^([24]); the amino acid sequence is as shown inSEQ ID No. 10; and the cDNA sequence encoding this amino acid sequenceis as shown in SEQ ID No. 9. Micro-plasminogen comprises only the serineprotease domain, the amino acid sequence of which has been reported inthe literature to comprise residues Ala543-Asn791 (with the Glu residueof the Glu-plasminogen sequence that does not contain a signal peptideas the starting amino acid)^([25]), and the sequence of which has beenalso reported in patent document CN 102154253 A to comprise residuesLys531-Asn791 (with the Glu residue of the Glu-plasminogen sequence thatdoes not contain a signal peptide as the starting amino acid) (thesequence in this patent application refers to the patent document CN102154253 A); the amino acid sequence is as shown in SEQ ID No. 12; andthe cDNA sequence encoding this amino acid sequence is as shown in SEQID No. 11.

In the present invention, “plasmin” is used interchangeably with“fibrinolysin” and “fibrinoclase”, and the terms have the same meaning;and “plasminogen” is used interchangeably with “profibrinolysin” and“fibrinoclase zymogen”, and the terms have the same meaning.

In the present application, the meaning of “lack” in plasminogen is thatthe content or activity of plasminogen in the body of a subject is lowerthan that of a normal person, which is low enough to affect the normalphysiological function of the subject; and the meaning of “deficiency”in plasminogen is that the content or activity of plasminogen in thebody of a subject is significantly lower than that of a normal person,or even the activity or expression is extremely small, and only throughexogenous supply can the normal physiological function be maintained.

Those skilled in the art can understand that all the technical solutionsof the plasminogen of the present invention are suitable for plasmin.Therefore, the technical solutions described in the present inventioncover plasminogen and plasmin.

In the course of circulation, plasminogen is in a closed, inactiveconformation, but when bound to thrombi or cell surfaces, it isconverted into an active plasmin in an open conformation under themediation of a plasminogen activator (PA). The active plasmin canfurther hydrolyze the fibrin clots to fibrin degradation products andD-dimers, thereby dissolving the thrombi. The PAp domain of plasminogencomprises an important determinant that maintains plasminogen in aninactive, closed conformation, and the KR domain is capable of bindingto lysine residues present on receptors and substrates. A variety ofenzymes that can serve as plasminogen activators are known, including:tissue plasminogen activator (tPA), urokinase plasminogen activator(uPA), kallikrein, coagulation factor XII (Hagmann factor), and thelike.

“Plasminogen active fragment” refers to an active fragment in theplasminogen protein that is capable of binding to a target sequence in asubstrate and exerting the proteolytic function. The technical solutionsof the present invention involving plasminogen encompass technicalsolutions in which plasminogen is replaced with a plasminogen activefragment. The plasminogen active fragment of the present invention is aprotein comprising a serine protease domain of plasminogen. Preferably,the plasminogen active fragment of the present invention comprises SEQID No.14, or an amino acid sequence having an amino acid sequenceidentity of at least 80%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ IDNo.14. Therefore, plasminogen of the present invention comprises aprotein containing the plasminogen active fragment and still having theplasminogen activity.

At present, methods for determining plasminogen and its activity inblood include: detection of tissue plasminogen activator activity(t-PAA), detection of tissue plasminogen activator antigen (t-PAAg) inplasma, detection of tissue plasminogen activity (plgA) in plasma,detection of tissue plasminogen antigen (plgAg) in plasma, detection ofactivity of the inhibitor of tissue plasminogen activators in plasma,detection of inhibitor antigens of tissue plasminogen activators inplasma and detection of plasmin-anti-plasmin (PAP) complex in plasma.The most commonly used detection method is the chromogenic substratemethod: streptokinase (SK) and a chromogenic substrate are added to atest plasma, the PLG in the test plasma is converted into PLM by theaction of SK, PLM acts on the chromogenic substrate, and then it isdetermined that the increase in absorbance is directly proportional toplasminogen activity using a spectrophotometer. In addition, plasminogenactivity in blood can also be determined by immunochemistry, gelelectrophoresis, immunonephelometry, radioimmuno-diffusion and the like.

“Orthologues or orthologs” refer to homologs between different species,including both protein homologs and DNA homologs, and are also known asorthologous homologs and vertical homologs. The term specifically refersto proteins or genes that have evolved from the same ancestral gene indifferent species. The plasminogen of the present invention includeshuman natural plasminogen, and also includes orthologues or orthologs ofplasminogens derived from different species and having plasminogenactivity.

“Conservatively substituted variant” refers to one in which a givenamino acid residue is changed without altering the overall conformationand function of the protein or enzyme, including, but not limited to,replacing an amino acid in the amino acid sequence of the parent proteinby an amino acid with similar properties (such as acidity, alkalinity,hydrophobicity, etc.). Amino acids with similar properties are wellknown. For example, arginine, histidine and lysine are hydrophilic basicamino acids and are interchangeable. Similarly, isoleucine is ahydrophobic amino acid that can be replaced by leucine, methionine orvaline. Therefore, the similarity of two proteins or amino acidsequences with similar functions may be different. For example, thesimilarity (identity) is 70%-99% based on the MEGALIGN algorithm.“Conservatively substituted variant” also includes a polypeptide orenzyme having amino acid identity of 60% or more, preferably 75% ormore, more preferably 85% or more, even more preferably 90% or more asdetermined by the BLAST or FASTA algorithm, and having the same orsubstantially similar properties or functions as the natural or parentprotein or enzyme.

“Isolated” plasminogen refers to the plasminogen protein that isisolated and/or recovered from its natural environment. In someembodiments, the plasminogen will be purified (1) to a purity of greaterthan 90%, greater than 95% or greater than 98% (by weight), asdetermined by the Lowry method, such as more than 99% (by weight); (2)to a degree sufficiently to obtain at least 15 residues of theN-terminal or internal amino acid sequence using a spinning cupsequenator; or (3) to homogeneity, which is determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing ornon-reducing conditions using Coomassie blue or silver staining.Isolated plasminogen also includes plasminogen prepared from recombinantcells by bioengineering techniques and separated by at least onepurification step.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein and refer to polymeric forms of amino acids ofany length, which may include genetically encoded and non-geneticallyencoded amino acids, chemically or biochemically modified or derivatizedamino acids, and polypeptides having modified peptide backbones. Theterm includes fusion proteins, including, but not limited to, fusionproteins having heterologous amino acid sequences, fusions havingheterologous and homologous leader sequences (with or without N-terminalmethionine residues); and the like.

The “percent amino acid sequence identity (%)” with respect to thereference polypeptide sequence is defined as the percentage of aminoacid residues in the candidate sequence identical to the amino acidresidues in the reference polypeptide sequence when a gap is introducedas necessary to achieve maximal percent sequence identity and noconservative substitutions are considered as part of sequence identity.The comparison for purposes of determining percent amino acid sequenceidentity can be achieved in a variety of ways within the skill in theart, for example using publicly available computer softwares, such asBLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for aligning sequences,including any algorithm needed to achieve the maximum comparison overthe full length of the sequences being compared. However, for purposesof the present invention, the percent amino acid sequence identity valueis generated using the sequence comparison computer program ALIGN-2.

In the case of comparing amino acid sequences using ALIGN-2, the % aminoacid sequence identity of a given amino acid sequence A relative to agiven amino acid sequence B (or may be expressed as a given amino acidsequence A having or containing a certain % amino acid sequence identityrelative to, with or for a given amino acid sequence B) is calculated asfollows:fraction X/Y×100

wherein X is the number of identically matched amino acid residuesscored by the sequence alignment program ALIGN-2 in the alignment of Aand B using the program, and wherein Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A relative to B will not be equal tothe % amino acid sequence identity of B relative to A. Unlessspecifically stated otherwise, all the % amino acid sequence identityvalues used herein are obtained using the ALIGN-2 computer program asdescribed in the previous paragraph.

As used herein, the terms “treatment” and “treating” refer to obtaininga desired pharmacological and/or physiologic effect. The effect may becomplete or partial prevention of a disease or its symptoms and/orpartial or complete cure of the disease and/or its symptoms, andincludes: (a) prevention of the disease from developing in a subjectthat may have a predisposition to the disease but has not been diagnosedas having the disease; (b) suppression of the disease, i.e., blockingits formation; and (c) alleviation of the disease and/or its symptoms,i.e., eliminating the disease and/or its symptoms.

The terms “individual”, “subject” and “patient” are used interchangeablyherein and refer to mammals, including, but not limited to, murine (ratsand mice), non-human primates, humans, dogs, cats, hoofed animals (e.g.,horses, cattle, sheep, pigs, goats) and so on.

“Therapeutically effective amount” or “effective amount” refers to anamount of plasminogen sufficient to achieve the prevention and/ortreatment of a disease when administered to a mammal or another subjectto treat the disease. The “therapeutically effective amount” will varydepending on the plasminogen used, the severity of the disease and/orits symptoms, as well as the age, body weight of the subject to betreated, and the like.

Preparation of the Plasminogen of the Present Invention

Plasminogen can be isolated and purified from nature for furthertherapeutic uses, and can also be synthesized by standard chemicalpeptide synthesis techniques. When chemically synthesized, a polypeptidecan be subjected to liquid or solid phase synthesis. Solid phasepolypeptide synthesis (SPPS) is a method suitable for chemical synthesisof plasminogen, in which the C-terminal amino acid of a sequence isattached to an insoluble support, followed by the sequential addition ofthe remaining amino acids in the sequence. Various forms of SPPS, suchas Fmoc and Boc, can be used to synthesize plasminogen. Techniques forsolid phase synthesis are described in Barany and Solid-Phase PeptideSynthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol.2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J.Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al. Solid Phase PeptideSynthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and GanesanA. 2006 Mini Rev. Med Chem. 6:3-10 and Camarero J A et al. 2005 ProteinPept Lett. 12:723-8. Briefly, small insoluble porous beads are treatedwith a functional unit on which a peptide chain is constructed. Afterrepeated cycles of coupling/deprotection, the attached solid phase freeN-terminal amine is coupled to a single N-protected amino acid unit.This unit is then deprotected to expose a new N-terminal amine that canbe attached to another amino acid. The peptide remains immobilized onthe solid phase before it is cut off.

Standard recombinant methods can be used to produce the plasminogen ofthe present invention. For example, a nucleic acid encoding plasminogenis inserted into an expression vector, so that it is operably linked toa regulatory sequence in the expression vector. Expression regulatorysequence includes, but is not limited to, promoters (e.g., naturallyassociated or heterologous promoters), signal sequences, enhancerelements and transcription termination sequences. Expression regulationcan be a eukaryotic promoter system in a vector that is capable oftransforming or transfecting eukaryotic host cells (e.g., COS or CHOcells). Once the vector is incorporated into a suitable host, the hostis maintained under conditions suitable for high-level expression of thenucleotide sequence and collection and purification of plasminogen.

A suitable expression vector is usually replicated in a host organism asan episome or as an integral part of the host chromosomal DNA. Ingeneral, an expression vector contains a selective marker (e.g.,ampicillin resistance, hygromycin resistance, tetracycline resistance,kanamycin resistance or neomycin resistance) to facilitate detection ofthose exogenous cells transformed with a desired DNA sequence.

Escherichia coli is an example of prokaryotic host cells that can beused to clone a polynucleotide encoding the subject antibody. Othermicrobial hosts suitable for use include Bacillus, for example, Bacillussubtilis and other species of enterobacteriaceae (such as Salmonellaspp. and Serratia spp.), and various Pseudomonas spp. In theseprokaryotic hosts, expression vectors can also be generated which willtypically contain an expression control sequence (e.g., origin ofreplication) that is compatible with the host cell. In addition, therewill be many well-known promoters, such as the lactose promoter system,the tryptophan (trp) promoter system, the beta-lactamase promoter systemor the promoter system from phage lambda. Optionally in the case ofmanipulation of a gene sequence, a promoter will usually controlexpression, and has a ribosome binding site sequence and the like toinitiate and complete transcription and translation.

Other microorganisms, such as yeast, can also be used for expression.Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitableyeast host cells, in which a suitable vector has an expression controlsequence (e.g., promoter), an origin of replication, a terminationsequence and the like, as required. A typical promoter comprises3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeastpromoters specifically include promoters derived from alcoholdehydrogenase, isocytochrome C, and enzymes responsible for maltose andgalactose utilization.

In addition to microorganisms, mammalian cells (e.g., mammalian cellscultured in cell culture in vitro) can also be used to express andgenerate the anti-Tau antibody of the present invention (e.g., apolynucleotide encoding a subject anti-Tau antibody). See Winnacker,From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). Suitablemammalian host cells include CHO cell lines, various Cos cell lines,HeLa cells, myeloma cell lines and transformed B cells or hybridomas.Expression vectors for these cells may comprise an expression controlsequence, such as an origin of replication, promoter and enhancer (Queenet al. Immunol. Rev. 89:49 (1986)), as well as necessary processinginformation sites, such as a ribosome binding site, RNA splice site,polyadenylation site and transcription terminator sequence. Examples ofsuitable expression control sequences are promoters derived from whiteimmunoglobulin gene, SV40, adenovirus, bovine papilloma virus,cytomegalovirus and the like. See Co et al. J. Immunol. 148:1149 (1992).

Once synthesized (chemically or recombinantly), the plasminogen of thepresent invention can be purified according to standard procedures inthe art, including ammonium sulfate precipitation, affinity column,column chromatography, high performance liquid chromatography (HPLC),gel electrophoresis and the like. The plasminogen is substantially pure,e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, atleast about 90% to 95% pure, or 98% to 99% pure or purer, for examplefree of contaminants such as cell debris, macromolecules other than thesubject antibody and the like.

Pharmaceutical Formulations

A therapeutic formulation can be prepared by mixing plasminogen of adesired purity with an optional pharmaceutical carrier, excipient orstabilizer (Remington's Pharmaceutical Sciences, 16th edition, Osol, A.ed. (1980)) to form a lyophilized preparation or an aqueous solution.Acceptable carriers, excipients and stabilizers are non-toxic to therecipient at the dosages and concentrations employed, and includebuffers, such as phosphates, citrates and other organic acids;antioxidants, including ascorbic acid and methionine; preservatives(e.g., octadecyl dimethyl benzyl ammonium chloride; hexane chloridediamine; benzalkonium chloride and benzethonium chloride; phenol,butanol or benzyl alcohol; alkyl p-hydroxybenzoates, such as methyl orpropyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol;3-pentanol; and m-cresol); low molecular weight polypeptides (less thanabout 10 residues); proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone;amino acids, such as glycine, glutamine, asparagine, histidine, arginineor lysine; monosaccharides, disaccharides and other carbohydrates,including glucose, mannose or dextrins; chelating agents, such as EDTA;sugars, such as sucrose, mannitol, fucose or sorbitol; salt-formingcounterions, such as sodium; metal complexes (e.g., zinc-proteincomplexes); and/or non-ionic surfactants, such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Preferred lyophilized anti-VEGF antibodyformulations are described in WO 97/04801, which is incorporated hereinby reference.

The formulations of the invention may also comprise one or more activecompounds required for the particular condition to be treated,preferably those that are complementary in activity and have no sideeffects with one another, for example anti-hypertensive drugs,anti-arrhythmic drugs, drugs for treating diabetes mellitus, and thelike.

The plasminogen of the present invention may be encapsulated inmicrocapsules prepared by techniques such as coacervation or interfacialpolymerization, for example, it may be incorporated in a colloid drugdelivery system (e.g., liposomes, albumin microspheres, microemulsions,nanoparticles and nanocapsules), or incorporated inhydroxymethylcellulose or gel-microcapsules and poly-(methylmethacrylate) microcapsules in macroemulsions. These techniques aredisclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.Ed. (1980).

The plasminogen of the present invention for in vivo administration mustbe sterile. This can be easily achieved by filtration through a sterilefiltration membrane before or after freeze drying and reconstitution.

The plasminogen of the present invention can be prepared into asustained-release preparation. Suitable examples of sustained-releasepreparations include solid hydrophobic polymer semi-permeable matriceshaving a shape and containing glycoproteins, such as films ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate)) (Langeret al. J. Biomed. Mater. Res., 15: 167-277 (1981); and Langer, Chem.Tech., 12:98-105 (1982)), or poly(vinyl alcohol), polylactides (U.S.Pat. No. 3,773,919, and EP 58,481), copolymer of L-glutamic acid and □ethyl-L-glutamic acid (Sidman et al. Biopolymers 22:547 (1983)),nondegradable ethylene-vinyl acetate (Langer et al. supra), ordegradable lactic acid-glycolic acid copolymers such as Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly D-(−)-3-hydroxybutyric acid. Polymers,such as ethylene-vinyl acetate and lactic acid-glycolic acid, are ableto persistently release molecules for 100 days or longer, while somehydrogels release proteins for a shorter period of time. A rationalstrategy for protein stabilization can be designed based on relevantmechanisms. For example, if the aggregation mechanism is discovered tobe formation of an intermolecular S—S bond through thio-disulfideinterchange, stability is achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

Administration and Dosage

The pharmaceutical composition of the present invention is administeredin different ways, for example by intravenous, intraperitoneal,subcutaneous, intracranial, intrathecal, intraarterial (e.g., viacarotid), and intramuscular administration.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, and alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, or fixed oils. Intravenousvehicles include liquid and nutrient supplements, electrolytesupplements and the like. Preservatives and other additives may also bepresent, for example, such as antimicrobial agents, antioxidants,chelating agents and inert gases.

The medical staff will determine the dosage regimen based on variousclinical factors. As is well known in the medical field, the dosage ofany patient depends on a variety of factors, including the patient'ssize, body surface area, age, the specific compound to be administered,sex, frequency and route of administration, overall health and otherdrugs administered simultaneously. The dosage range of thepharmaceutical composition comprising plasminogen of the presentinvention may be, for example, such as about 0.0001 to 2000 mg/kg, orabout 0.001 to 500 mg/kg (such as 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg,0.75 mg/kg, 10 mg/kg and 50 mg/kg) of the subject's body weight daily.For example, the dosage may be 1 mg/kg body weight or 50 mg/kg bodyweight, or in the range of 1 mg/kg-50 mg/kg, or at least 1 mg/kg.Dosages above or below this exemplary range are also contemplated,especially considering the above factors. The intermediate dosages inthe above range are also included in the scope of the present invention.A subject may be administered with such dosages daily, every other day,weekly or based on any other schedule determined by empirical analysis.An exemplary dosage schedule includes 1-10 mg/kg for consecutive days.During administration of the drug of the present invention, thetherapeutic effect and safety are required to be assessed real-timely.

Articles of Manufacture or Kits

One embodiment of the present invention relates to an article ofmanufacture or a kit comprising plasminogen of the present invention orplasmin useful in the treatment of angiocardiopathy and its relatedconditions caused by diabetes mellitus. The article preferably includesa container, label or package insert. Suitable containers includebottles, vials, syringes and the like. The container can be made ofvarious materials, such as glass or plastic. The container contains acomposition that is effective to treat the disease or condition of thepresent invention and has a sterile access (for example, the containermay be an intravenous solution bag or vial containing a plug that can bepierced by a hypodermic injection needle). At least one active agent inthe composition is plasminogen/plasmin. The label on or attached to thecontainer indicates that the composition is used to treat theangiocardiopathy and its related conditions caused by diabetes mellitusaccording to the present invention. The article may further comprise asecond container containing a pharmaceutically acceptable buffer, suchas phosphate buffered saline, Ringer's solution and glucose solution. Itmay further comprise other substances required from a commercial anduser perspective, including other buffers, diluents, filters, needlesand syringes. In addition, the article comprises a package insert withinstructions for use, including, for example, instructions to direct auser of the composition to administer to a patient the plasminogencomposition and other drugs for treating an accompanying disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows observed results of Sirius red staining of liver afteradministration of plasminogen to carbon tetrachloride-induced hepaticfibrosis model mice for 28 days. A represents the blank control group, Brepresents the control group administered with vehicle PBS, C representsthe group administered with plasminogen, and D represents thequantitative analysis results. The results showed that the collagendeposition (indicated by arrow) in the group administered withplasminogen was remarkably less than that in the control groupadministered with vehicle PBS, and the statistical difference wassignificant (* indicates P<0.05); compared with the control groupadministered with vehicle PBS, the level of collagen deposition in micein the group administered with plasminogen was closer to that in blankcontrol mice. It indicates that plasminogen can reduce collagendeposition in liver, and ameliorate hepatic fibrosis in hepatic fibrosismodel mice.

FIG. 2 shows a representative image of Sirius red staining of aorticsinus after administration of plasminogen to ApoE atherosclerosis modelmice for 30 days. A and C refer to the control group administered withvehicle PBS, and B and D refer to the group administered withplasminogen. The results showed that the area of collagen deposition(indicated by arrow) in the group administered with plasminogen wasremarkably less than that in the control group administered with vehiclePBS, indicating that plasminogen can reduce the level of aortic sinusfibrosis in arteriosclerosis model mice.

FIG. 3 shows a representative image of Sirius red staining of liverafter administration of plasminogen to carbon tetrachloride-inducedhepatic fibrosis model mice for 14 days. A represents a blank controlgroup, B represents a control group administered with vehicle PBS, and Crepresents a group administered with plasminogen. The results showedthat in the group administered with plasminogen, the collagen depositionwas remarkably less than that in the control group administered withvehicle PBS, and the level of collagen deposition in mice was close tothat in blank control mice. It indicates that plasminogen can reducecollagen deposition in liver, and ameliorate hepatic fibrosis in hepaticfibrosis model mice.

FIG. 4 shows a representative image of Sirius red staining of aorticsinus after administration of plasminogen to 16-week hyperlipemia modelmice for 30 days. A and C refer to the control group administered withvehicle PBS, and B and D refer to the group administered withplasminogen. The results showed that the area of collagen deposition(indicated by arrow) on the intima of the aortic sinus wall in the groupadministered with plasminogen was remarkably less than that in thecontrol group administered with vehicle PBS, indicating that plasminogencan reduce the level of intimal fibrosis of the aortic sinus wall inhyperlipemia model mice.

FIG. 5 shows a representative image of Sirius red staining of skin afteradministration of plasminogen to bleomycin-induced systemic sclerosismodel mice for 21 days. A represents the blank control group, Brepresents the control group administered with vehicle PBS, C representsthe group administered with plasminogen, and D represents the group withimpaired PLG activity. The results showed that in the bleomycin-inducedsystemic sclerosis mouse model, the collagen fiber bundles in the upperdermis were remarkably increased, the collagen fibers were thick andbig, and dense in arrangement, and the dermal layer was thickened in thegroup administered with vehicle PBS and the group with impaired PLGactivity; while in the group administered with plasminogen, thefibroblasts in the dermal layer were remarkably less than those in thegroup administered with vehicle PBS, and the thickness of the dermallayer of the skin was close to the normal level.

FIG. 6 shows a representative image of Sirius red staining of lung afteradministration of plasminogen to bleomycin-induced systemic sclerosismodel mice for 21 days. A represents the control group administered withvehicle PBS, B represents the group administered with plasminogen, and Crepresents the quantitative analysis results. The results showed that inthe bleomycin-induced systemic sclerosis mouse model, the degree ofpulmonary fibrosis (indicated by arrow) in mice in the groupadministered with vehicle PBS was higher than that in the groupadministered with plasminogen; while in the group administered withplasminogen, the morphology of pulmonary alveolar walls in mice wasclose to normal, cells with the inflammatory level was remarkablyreduced, the degree of fibrosis was remarkably lower than that in thegroup administered with vehicle PBS, and the statistical difference wassignificant (* indicates P<0.05).

FIG. 7 shows a representative image of Sirius red staining of heartafter administration of plasminogen to bleomycin-induced systemicsclerosis model mice for 21 days. A represents the control groupadministered with vehicle PBS, and B represents the group administeredwith plasminogen. Studies have found that in the bleomycin-inducedsystemic sclerosis mouse model, the degree of collagen deposition(indicated by arrow) in heart in the control group administered withvehicle PBS was higher than that in the group administered withplasminogen. It indicates that plasminogen can effectively reducebleomycin-induced cardiac fibrosis.

FIG. 8 shows a representative image of Sirius red staining of kidneyafter administration of plasminogen to bleomycin-induced systemicsclerosis model mice for 21 days. A represents the control groupadministered with vehicle PBS, and B represents the group administeredwith plasminogen. The results showed that in the bleomycin-inducedsystemic sclerosis mouse model, the degree of collagen fibrosis(indicated by arrow) in the kidney in the control group administeredwith vehicle PBS was higher than that in the group administered withplasminogen. It indicates that plasminogen can effectively reducebleomycin-induced renal fibrosis.

FIG. 9 shows observed results of immunostaining of type IV collagen inthe kidney after administration of plasminogen to 24- to 25-week-olddiabetic mice for 31 days. A represents the control group administeredwith vehicle PBS, and B represents the group administered withplasminogen. The results showed that the positive staining (indicated byarrow) of IV collagen in the group administered with plasminogen wasremarkably higher than that in the control group administered withvehicle PBS, indicating that plasminogen can ameliorate renal fibrosisin diabetic mice.

FIG. 10 shows observed results of masson staining of kidney afteradministration of plasminogen to 26-week-old diabetic mice for 35 days.A represents the control group administered with vehicle PBS, and Brepresents the group administered with plasminogen. The results showedthat in the control group administered with vehicle PBS, glomerularmesangial hyperplasia existed, mesangial matrix increased, renalinterstitial fibrosis was mild (indicated by arrow), and thehyperplastic fibrosis was blue. In the group administered withplasminogen, the glomerular mesangial cells and matrix were remarkablyless than those in the control group, and renal interstitial fibrosiswas remarkably reduced. It indicates that plasminogen can amelioratefibrotic lesions in the kidneys of diabetic mice.

FIG. 11 shows observed results of masson staining of heart afteradministration of plasminogen to 24- to 25-week-old diabetic mice for 31days. A represents the control group administered with vehicle PBS, andB represents the group administered with plasminogen. The results showedthat in the control group administered with vehicle PBS, bluehyperplastic collagen fibers (indicated by arrow) could be seen betweenmyocardial fibers, showing mild myocardial fibrosis; while in the groupadministered with plasminogen, a few light blue hyperplastic collagenfibers could be seen between myocardial fibers, and the myocardialfibrosis was remarkably alleviated compared with the control group. Itindicates that plasminogen can ameliorate cardiac fibrosis in diabeticmice.

FIG. 12 shows a representative image of Sirius red staining of heartafter administration of plasminogen to 17- to 18-week-old diabetic micefor 35 days. A represents the control group administered with vehiclePBS, and B represents the group administered with plasminogen. Theresults showed that the deposition of collagen fibers (indicated byarrow) in mice in the group administered with plasminogen was remarkablyless than that in the control group administered with vehicle PBS. Itindicates that plasminogen can reduce cardiac fibrosis in relativelyyoung (17- to 18-week-old) diabetic mice.

FIG. 13 shows a representative image of Sirius red staining of heartafter administration of plasminogen to 26- to 27-week-old diabetic micefor 35 days. A represents the control group administered with vehiclePBS, and B represents the group administered with plasminogen. Theresults showed that the collagen deposition (indicated by arrow) in micein the group administered with plasminogen was remarkably less than thatin the control group administered with vehicle PBS. It indicates thatplasminogen can attenuate cardiac fibrosis in relatively old (26- to27-week-old) diabetic mice.

FIG. 14 shows observed results of immunostaining of type IV collagen inthe kidney after administration of plasminogen to cisplatin-inducedrenal fibrosis model mice for 7 days. A represents the control groupadministered with vehicle PBS, and B represents the group administeredwith plasminogen. The results showed that the positive expression(indicated by arrow) of type IV collagen in the kidney in the controlgroup administered with vehicle PBS was remarkably higher than that inthe group administered with plasminogen. It indicates that plasminogencan ameliorate renal fibrosis in cisplatin-induced renal fibrosis modelmice.

FIG. 15 shows a representative image of Sirius red staining of heartafter administration of plasminogen to ApoE atherosclerosis model micefor 30 days. A represents the control group administered with vehiclePBS, and B represents the group administered with plasminogen. Theresults showed that the collagen deposition (indicated by arrow) in thegroup administered with plasminogen was remarkably less than that in thecontrol group administered with vehicle PBS, indicating that plasminogencan alleviate cardiac fibrosis in ApoE atherosclerosis model mice.

FIG. 16 shows a representative image of Sirius red staining of heartafter administration of plasminogen to C57 hyperlipemia model mice for30 days. A represents the control group administered with vehicle PBS,and B represents the group administered with plasminogen. The resultsshowed that the collagen deposition (indicated by arrow) in the groupadministered with plasminogen was remarkably less than that in thecontrol group administered with vehicle PBS, indicating that plasminogencan alleviate cardiac fibrosis in hyperlipemia model mice.

FIG. 17 shows observed results of Sirius red staining of kidney afteradministration of plasminogen to purine-induced chronic renal injurymodel mice for 10 days. A represents the control group administered withvehicle PBS, B represents the group administered with plasminogen, Crepresents the group with impaired PLG activity, and D represents thequantitative analysis results. The collagen deposition (indicated byarrow) in the group administered with plasminogen was remarkably lessthan that in the control group administered with vehicle PBS and thegroup with impaired PLG activity, and the quantitative analysis showed asignificant statistical difference between the group administered withplasminogen and the group with impaired PLG activity (* indicatesP<0.05). It indicates that plasminogen can alleviate renal fibrosisinduced by chronic renal injury, and promote the repair of renal injury.

FIG. 18 shows the observed results of Sirius red-staining for pancreaticislets after administration of plasminogen to 24- to 25-week-olddiabetic mice for 31 days. A represents the control group administeredwith vehicle PBS, B represents the group administered with plasminogen,and C represents the quantitative analysis results. The results showedthat the collagen deposition (indicated by arrow) in the pancreaticislet of mice in the group administered with plasminogen was remarkablyless than that in the control group administered with vehicle PBS, andthe statistical difference was significant (* indicates P<0.05). Itindicates that plasminogen can ameliorate injury and fibrosis of thepancreatic islet caused by diabetes mellitus.

FIG. 19 shows observed results of Sirius red staining of kidney afteradministration of plasminogen to 3% cholesterol hyperlipemia model micefor 30 days. A represents the blank control group, B represents thecontrol group administered with vehicle PBS, C represents the groupadministered with plasminogen, and D represents the quantitativeanalysis results. The results showed that the collagen deposition inkidney (indicated by arrow) in the group administered with plasminogenwas remarkably less than that in the control group administered withvehicle PBS, and the statistical difference was significant; and in thegroup administered with plasminogen, fibrosis was substantially restoredto a normal level. It indicates that plasminogen can effectively reducerenal fibrosis in 3% cholesterol hyperlipemia model mice.

EXAMPLES Example 1. Plasminogen Reduces Collagen Deposition in LiverDuring Induction of Hepatic Fibrosis by Carbon Tetrachloride

Twenty 7- to 8-week-old female C57 mice were randomly divided into threegroups, 5 mice in the blank control group, 7 mice the control groupadministered with vehicle PBS, and 8 mice in the group administered withplasminogen. Mice in the control group administered with vehicle PBS andthe group administered with plasminogen were injected with carbontetrachloride intraperitoneally at a dose of 1 mL/kg body weight, threetimes a week for four consecutive weeks, to establish the hepaticfibrosis model^([36,37]); while the blank control mice were injectedwith a corresponding volume of corn oil intraperitoneally. Carbontetrachloride required to be diluted with corn oil, and the dilutionratio of carbon tetrachloride to corn oil was 1:3. Administration beganon the day of model establishment, i.e., Day 1. Mice in the groupadministered with plasminogen were injected with human plasminogen at adose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS via the tail vein, both lasting for 28 days. The blankcontrol group was not treated with injection. The mice were sacrificedon Day 29. The livers were fixed in 4% paraformaldehyde for 24 hours.The fixed livers were paraffin-embedded after dehydration with alcoholgradient and permeabilization with xylene. The tissue sections was 3 μmthick. The sections were dewaxed and rehydrated and washed with wateronce. After stained with 0.1% Sirius red for 60 min, the sections wereflushed with running water. After stained with hematoxylin for 1 min,the sections were flushed with running water, differentiated with 1%hydrochloric acid in alcohol and returned to blue with ammonia water,flushed with running water, dried and sealed. The sections were observedunder an optical microscope at 200×.

The results showed that the collagen deposition in the groupadministered with plasminogen (FIG. 1C) was remarkably less than that inthe control group administered with vehicle PBS (FIG. 1B), and thestatistical difference was significant (FIG. 1D); compared with thecontrol group administered with vehicle PBS, the level of collagendeposition (indicated by arrow) in mice in the group administered withplasminogen was closer to that in blank control mice (FIG. 1A). Itindicates that plasminogen can reduce collagen deposition in liver, andameliorate hepatic fibrosis in hepatic fibrosis model mice.

Example 2. Plasminogen Ameliorates Aortic Sinus Fibrosis in ApoEAtherosclerosis Mice

Thirteen 6-week-old male ApoE mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe atherosclerosis model^([31,32]). 50 μL of blood was taken from eachmodel mouse three days before administration, and the total cholesterol(T-CHO) content was detected. The mice were randomly divided into twogroups based on the T-CHO content, 7 mice in the control groupadministered with vehicle PBS, and 6 mice in the group administered withplasminogen. The first day of administration was set as Day 1. Mice inthe group administered with plasminogen were injected with humanplasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and anequal volume of PBS was administered to mice in the control groupadministered with vehicle PBS via the tail vein. The mice wereadministered for 30 days and sacrificed on Day 31. The hearts were fixedin 4% paraformaldehyde for 24 to 48 hours, then sedimented in 15% and30% sucrose at 4° C. overnight, respectively, and embedded in OCT. Thefrozen sections were 8 μm thick. After stained with 0.1% Sirius red insaturated picric acid for 30 min, the sections were flushed with runningwater for 2 min. After stained with hematoxylin for 1 min, the sectionswere flushed with running water, differentiated with 1% hydrochloricacid in alcohol, returned to blue with ammonia water, flushed withrunning water, dried and sealed with a neutral gum. The sections wereobserved under an optical microscope at 40× (FIGS. 2A and 2B) and 200×(FIGS. 2C and 2D).

The results showed that the area of collagen deposition (indicated byarrow) on the intima of the aortic sinus wall in the group administeredwith plasminogen (FIGS. 2B and D) was remarkably less than that in thecontrol group administered with vehicle PBS (FIGS. 2A and C), indicatingthat plasminogen can reduce the level of aortic sinus fibrosis inarteriosclerosis model mice.

Example 3. Plasminogen Ameliorates Carbon Tetrachloride-Induced HepaticFibrosis

Fifteen 9-week-old female C57 mice were randomly divided into threegroups, a blank control group, a control group administered with vehiclePBS, and a group administered with plasminogen, 5 mice in each group.Mice in the control group administered with vehicle PBS and the groupadministered with plasminogen were injected with carbon tetrachlorideintraperitoneally at a dose of 1 mL/kg body weight, three times a weekfor two consecutive weeks, to establish the hepatic fibrosismodel^([36,37]); while the blank control mice were injected with acorresponding volume of corn oil according to the injection method ofmodel mice.

Carbon tetrachloride required to be diluted with corn oil, and thedilution ratio of carbon tetrachloride to corn oil was 1:3.Administration began after model establishment. The first day ofadministration was recorded as Day 1. Mice in the group administeredwith plasminogen were injected with human plasminogen at a dose of 1mg/0.1 mL/mouse/day via the tail vein, and mice in the control groupadministered with vehicle PBS were injected with an equal volume of PBSvia the tail vein, both lasting for 14 consecutive days. The blankcontrol group was not treated with injection. The mice were sacrificedon Day 15. The livers were fixed in 4% paraformaldehyde for 24 hours.The fixed livers were paraffin-embedded after dehydration with alcoholgradient and permeabilization with xylene. The tissue sections was 3 μmthick. The sections were dewaxed and rehydrated and washed with wateronce. After stained with 0.1% Sirius red for 60 min, the sections wereflushed with running water. After stained with hematoxylin for 1 min,the sections were flushed with running water, differentiated with 1%hydrochloric acid in alcohol and returned to blue with ammonia water,flushed with running water, dried and sealed. The sections were observedunder an optical microscope at 200×.

The results showed that the collagen deposition in the groupadministered with plasminogen (FIG. 3C) was remarkably less than that inthe control group administered with vehicle PBS (FIG. 3B), and comparedto the group administered with PBS, the level of collagen deposition inmice in the group administered with plasminogen was closer to that inblank control mice (FIG. 3A). It indicates that plasminogen can reducecollagen deposition in liver, and ameliorate hepatic fibrosis in hepaticfibrosis model mice.

Example 4. Plasminogen Reduces Aortic Sinus Fibrosis in 16-WeekHyperlipemia Model Mice

Eleven 6-week-old male C57 mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe hyperlipemia model^([30,31]). This model was designated as the16-week hyperlipemia model. The model mice continued to be fed with ahigh-cholesterol diet. 50 μL of blood was taken from each mouse threedays before administration, and the total cholesterol (T-CHO) contentwas detected. The mice were randomly divided into two groups based onthe T-CHO content, 6 mice in the control group administered with vehiclePBS, and 5 mice in the group administered with plasminogen. The firstday of administration was recorded as Day 1. Mice in the groupadministered with plasminogen were injected with human plasminogen at adose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS via the tail vein. The mice were administered for 30 daysand sacrificed on Day 31. The heart materials were taken and fixed in 4%paraformaldehyde for 24 to 48 hours. The fixed tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The aortic sinus sections was 3 μm thick.The sections were dewaxed and rehydrated and washed with water once.After stained with 0.1% Sirius red in saturated picric acid for 30 min,the sections were flushed with running water for 2 min. After stainedwith hematoxylin for 1 min, the sections were flushed with runningwater, differentiated with 1% hydrochloric acid in alcohol, returned toblue with ammonia water, flushed with running water, dried and sealedwith a neutral gum. The sections were observed under an opticalmicroscope at 40× (FIGS. 4A and 4B) and 200× (FIGS. 4C and 4D).

The results showed that the area of collagen deposition (indicated byarrow) on the intima of the aortic sinus wall in the group administeredwith plasminogen (FIGS. 4B and 4D) was remarkably less than that in thecontrol group administered with vehicle PBS (FIGS. 4A and 4C),indicating that plasminogen can alleviate the level of aortic sinusfibrosis in hyperlipemia model mice.

Example 5. Plasminogen Lowers Skin Fibrosis in Systemic Sclerosis Mice

Fifteen 12-week-old male C57 mice were randomly divided into threegroups, a blank control group, a control group administered with vehiclePBS (PBS refers to Phosphate Buffer Saline, as a vehicle of plasminogenherein), and a group administered with plasminogen, 5 mice in eachgroup, and five 13-week-old mice with impaired PLG activity were taken.The mice were weighed and grouped on the day when the experiment began,i.e., Day 0. Model establishment and administration began from the nextday, wherein mice in the control group administered with vehicle PBS andthe group administered with plasminogen as well as mice with impairedPLG activity were injected with bleomycin subcutaneously at a dose of0.1 mg/0.1 mL/mouse/day to induce systemic sclerosis^([26]). Mice in theblank control group were injected with normal saline subcutaneously at adose of 0.1 mL/mouse/day; meanwhile, on Day 1, plasminogen or PBS wasadministered for 21 consecutive days for model establishment. Mice inthe group administered with plasminogen were injected with plasminogenat a dose of 1 mg/0.1 mL/mouse/day via the tail vein, an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS, and the normal mouse group and mice with impaired PLGactivity were not treated. The mice were sacrificed on Day 22. The backskin tissues were fixed in 4% paraformaldehyde fixative for 24 hours.The fixed skin tissues were paraffin-embedded after dehydration withalcohol gradient and permeabilization with xylene. The tissue sectionswas 3 μm thick. The sections were dewaxed and rehydrated and washed withwater once. After stained with 0.1% Sirius red in saturated picric acidfor 30 min, the sections were flushed with running water for 2 min.After stained with hematoxylin for 1 min, the sections were flushed withrunning water, differentiated with 1% hydrochloric acid in alcohol,returned to blue with ammonia water, flushed with running water, driedand sealed with a neutral gum. The sections were observed under anoptical microscope at 100×.

Sirius red staining allows for long-lasting staining of collagen. As aspecial staining method for pathological sections, Sirius red stainingcan show the collagen tissue specifically.

The results showed that in the bleomycin-induced systemic sclerosismouse model, it was observed under a microscope that the collagen fiberbundles in the upper dermis were remarkably increased, the collagenfibers were thick and big, and dense in arrangement, and the dermallayer was thickened in mice in the group administered with vehicle PBS(FIG. 5B) and the group with impaired PLG activity (FIG. 5D); while inthe group administered with plasminogen (FIG. 5C), the fibroblasts inthe dermal layer were remarkably less than those in the control groupadministered with vehicle PBS, and the thickness of the dermal layer ofthe skin was substantially close to the normal level (FIG. 5A). Itindicates that plasminogen can effectively reduce bleomycin-induced skinfibrosis.

Example 6. Plasminogen Lowers Pulmonary Fibrosis in Systemic SclerosisMice

Seventeen 12-week-old male C57 mice were randomly divided into twogroups, 11 mice in the control group administered with vehicle PBS, and6 mice in the group administered with plasminogen. The mice were weighedand grouped on the day when the experiment began, i.e., Day 0. Modelestablishment and administration began from Day 1, wherein mice in bothgroups were injected with bleomycin subcutaneously at a dose of 0.1mg/0.1 mL/mouse/day to induce systemic sclerosis^([26]), and plasminogenor PBS was administered for 21 consecutive days for model establishment.Mice in the group administered with plasminogen were injected withplasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and anequal volume of PBS was administered to mice in the control groupadministered with vehicle PBS in the same manner. The mice weresacrificed on Day 22. The lung tissues were fixed in 4% paraformaldehydefixative for 24 hours. The fixed lung tissues were paraffin-embeddedafter dehydration with alcohol gradient and permeabilization withxylene. The tissue sections was 3 μm thick. The sections were dewaxedand rehydrated and washed with water once. After stained with 0.1%Sirius red in saturated picric acid for 30 min, the sections wereflushed with running water for 2 min. After stained with hematoxylin for1 min, the sections were flushed with running water, differentiated with1% hydrochloric acid in alcohol, returned to blue with ammonia water,flushed with running water, dried and sealed with a neutral gum. Thesections were observed under an optical microscope at 200×.

Studies have found that in the bleomycin-induced systemic sclerosismouse model, it was observed under a microscope that the degree ofcollagen fibrosis (indicated by arrow) in the group administered withvehicle PBS (FIG. 6A) was higher than that in the group administeredwith plasminogen (FIG. 6B); while in the group administered withplasminogen, the morphology of pulmonary alveolar walls in mice wasclose to the normal level, the inflammatory cells were remarkablyreduced, the degree of fibrosis was remarkably lower than that in thegroup administered with vehicle PBS, and the statistical difference wassignificant (FIG. 6C). It indicates that plasminogen can effectivelyreduce lung tissue fibrosis in bleomycin-induced systemic sclerosismice.

Example 7. Plasminogen Lowers Cardiac Fibrosis in Systemic SclerosisMice

Ten 12-week-old male C57 mice were randomly divided into two groups, 5mice in each of the control group administered with vehicle PBS and thegroup administered with plasminogen. The mice were weighed and groupedon the day when the experiment began, i.e., Day 0. Model establishmentand administration began from Day 1, wherein mice were injected withbleomycin subcutaneously at a dose of 0.1 mg/0.1 mL/mouse/day to inducesystemic sclerosis^([26]), and plasminogen or PBS was administered for21 consecutive days. Mice in the group administered with plasminogenwere injected with plasminogen at a dose of 1 mg/0.1 mL/mouse/day viathe tail vein, and an equal volume of PBS was administered to mice inthe control group administered with vehicle PBS via the tail vein. Themice were sacrificed on Day 22. The hearts were fixed in 4%paraformaldehyde fixative for 24 hours. The fixed hearts wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The tissue sections was 3 μm thick. Thesections were dewaxed and rehydrated and washed with water once. Afterstained with 0.1% Sirius red in saturated picric acid for 30 min, thesections were flushed with running water for 2 min. After stained withhematoxylin for 1 min, the sections were flushed with running water,differentiated with 1% hydrochloric acid in alcohol, returned to bluewith ammonia water, flushed with running water, dried and sealed with aneutral gum. The sections were observed under an optical microscope at200×.

Studies have found that in the bleomycin-induced systemic sclerosismouse model, it was observed under a microscope that the collagendeposition in heart in the control group administered with vehicle PBS(FIG. 7A) was higher than that in the group administered withplasminogen (FIG. 7B). It indicates that plasminogen can effectivelyreduce bleomycin-induced cardiac fibrosis.

Example 8. Plasminogen Lowers Renal Fibrosis in Systemic Sclerosis Mice

Ten 12-week-old male C57 mice were randomly divided into two groups, 5mice in each of the control group administered with vehicle PBS and thegroup administered with plasminogen. The mice were weighed and groupedon the day when the experiment began, i.e., Day 0. Model establishmentand administration began from Day 1, wherein all mice were injected withbleomycin subcutaneously at a dose of 0.1 mg/0.1 mL/mouse/day to inducesystemic sclerosis, and plasminogen or PBS was administered for 21consecutive days for model establishment. Mice in the group administeredwith plasminogen were injected with plasminogen at a dose of 1 mg/0.1mL/mouse/day via the tail vein, and mice in the control groupadministered with vehicle PBS were injected with an equal volume of PBSvia the tail vein. The mice were sacrificed on Day 22. The kidneys werefixed in 4% paraformaldehyde fixative for 24 hours. The fixed kidneyswere paraffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The tissue sections was 3 μm thick. Thesections were dewaxed and rehydrated and washed with water once. Afterstained with 0.1% Sirius red in saturated picric acid for 30 min, thesections were flushed with running water for 2 min. After stained withhematoxylin for 1 min, the sections were flushed with running water,differentiated with 1% hydrochloric acid in alcohol, returned to bluewith ammonia water, flushed with running water, dried and sealed with aneutral gum. The sections were observed under an optical microscope at200×.

The results showed that in the bleomycin-induced systemic sclerosismouse model, the degree of collagen fibrosis (indicated by arrow) in thekidney in the control group administered with vehicle PBS (FIG. 8A) washigher than that in the group administered with plasminogen (FIG. 8B).It indicates that plasminogen can effectively reduce bleomycin-inducedrenal fibrosis.

Example 9. Plasminogen Lowers Collagen Deposition in Kidney of DiabeticMice

Ten 24- to 25-week-old male db/db mice were randomly divided into twogroups, five mice in each of a control group administered with vehiclePBS and a group administered with plasminogen. The mice were weighed andgrouped on the day when the experiment began, i.e. Day 0. Plasminogen orPBS was administered from day 1 for 31 consecutive days. Mice in thegroup administered with plasminogen were injected with plasminogen at adose of 2 mg/0.2 mL/mouse/day via the tail vein, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS. The mice were sacrificed after administration ofplasminogen for 31 days. The kidney tissues were fixed in 4%paraformaldehyde fixative for 24 hours. The fixed kidney tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 4μm. The sections were dewaxed and rehydrated and washed with water once.The sections were incubated with 3% hydrogen peroxide for 15 minutes andwashed with 0.01M PBS twice for 5 minutes each time. The sections wereblocked with 10% normal goat serum (Vector laboratories, Inc., USA) for1 hour, and after the time was up, the serum was thrown away, and thetissues were circled with a PAP pen. The sections were incubated withrabbit anti-mouse polyclonal antibody (Abcam) against IV collagenovernight at 4° C. and washed with TBS twice for 5 minutes each time.The sections were incubated with a secondary antibody, goat anti-rabbitIgG (HRP) antibody (Abcam), for 1 hour at room temperature and washedwith TBS twice. The sections were developed with a DAB kit (Vectorlaboratories, Inc., USA). After washed with water three times, thesections were counterstained with hematoxylin for 30 seconds and flushedwith running water for 5 minutes. After dehydration with alcoholgradient, permeabilization with xylene, and sealing with a neutral gum,the sections were observed under an optical microscope at 200×.

Diabetic nephropathy is a chronic complication of diabetes mellitus, andglomerular sclerosis and renal interstitial fibrosis are typicalpathological changes^([27]).

The results showed that the positive staining of IV collagen in thegroup administered with plasminogen (FIG. 9B) was remarkably more thanthat in the control group administered with vehicle PBS (FIG. 9A),indicating that plasminogen can reduce collagen deposition (indicated byarrow) in the kidney tissue, and suggesting that plasminogen is expectedto prevent renal tissue fibrosis caused by diabetes mellitus by reducingcollagen deposition in the kidney tissue.

Example 10. Plasminogen Ameliorates Renal Fibrosis in Diabetic Mice

Ten 26-week-old male db/db mice were randomly divided into two groups, 5mice in each of the control group administered with vehicle PBS and thegroup administered with plasminogen. The mice were weighed and groupedon the day when the experiment began, i.e. Day 0. Plasminogen or PBS wasadministered from day 1 for 35 consecutive days. Mice in the groupadministered with plasminogen were injected with plasminogen at a doseof 2 mg/0.2 mL/mouse/day via the tail vein, and an equal volume of PBSwas administered to mice in the control group administered with vehiclePBS. The mice were sacrificed on Day 36. The kidney tissues were fixedin 4% paraformaldehyde fixative for 24 hours. The fixed kidney tissueswere paraffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 4μm. The sections were dewaxed and rehydrated and then put into apotassium dichromate solution overnight. The sections were stained withiron hematocylin for 3 to 5 minutes, and flushed slightly with runningwater. The sections were differentiated with 1% hydrochloric acid inalcohol, treated with ammonia water for 1 second, and rinsed with water.The sections were stained in ponceau acid fuchsin fluid for 8 minutes,and rinsed rapidly in water. The sections were treated with 1%phosphomolybdic acid aqueous solution for about 2 minutes, andcounterstained with aniline blue solution for 6 minutes. The sectionswere rinsed with 1% glacial acetic acid for about 1 minute. The sectionswere sealed after dehydration with absolute ethanol, andpermeabilization with xylene, and were observed under an opticalmicroscope at 200×.

Masson staining can reveal tissue fibrosis. The results showed that inthe control group administered with vehicle PBS (FIG. 10A), glomerularmesangial hyperplasia existed, mesangial matrix increased, renalinterstitial fibrosis was mild (indicated by arrow), and thehyperplastic fibrosis was blue. In the group administered withplasminogen (FIG. 10B), the glomerular mesangial cells and matrix wereremarkably less than those in the control group, and renal interstitialfibrosis was remarkably reduced. It indicates that plasminogen canameliorate renal fibrosis in diabetic mice.

Example 11. Plasminogen Ameliorates Cardiac Fibrosis in 24- to25-Week-Old Diabetic Mice

Ten 24- to 25-week-old male db/db mice were randomly divided into twogroups, five mice in each of a control group administered with vehiclePBS and a group administered with plasminogen. The mice were weighed andgrouped on the day when the experiment began, i.e. Day 0. Plasminogen orPBS was administered from day 1 for 31 consecutive days. Mice in thegroup administered with plasminogen were injected with plasminogen at adose of 2 mg/0.2 mL/mouse/day via the tail vein, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS. The mice were sacrificed after administration ofplasminogen for 31 days. The heart tissues were fixed in 4%paraformaldehyde fixative for 24 hours. The fixed heart tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 4μm. The sections were dewaxed and rehydrated and then put into apotassium dichromate solution overnight. The sections were stained withiron hematocylin for 3 to 5 minutes, and flushed slightly with runningwater. The sections were differentiated with 1% hydrochloric acid inalcohol, treated with ammonia water for 1 second, and rinsed with water.The sections were stained in ponceau acid fuchsin fluid for 8 minutes,and rinsed rapidly in water. The sections were treated with 1%phosphomolybdic acid aqueous solution for about 2 minutes, andcounterstained with aniline blue solution for 6 minutes. The sectionswere rinsed with 1% glacial acetic acid for about 1 minute. The sectionswere sealed after dehydration with absolute ethanol, andpermeabilization with xylene, and were observed under an opticalmicroscope at 200×.

The most common complication of diabetes mellitus is excessiveaccumulation of connective tissues (pathological fibrosis). Myocardialinterstitial fibrosis may be the characteristic pathological change ofdiabetic cardiomyopathy^([28-29]).

Masson staining can reveal tissue fibrosis. The results showed that inthe control group administered with vehicle PBS (FIG. 11A), bluehyperplastic collagen fibers (indicated by arrow) could be seen betweenmyocardial fibers, showing mild myocardial fibrosis; while in the groupadministered with plasminogen (FIG. 11B), a few light blue hyperplasticcollagen fibers could be seen between myocardial fibers, and themyocardial fibrosis was remarkably alleviated compared with the controlgroup. It indicates that plasminogen can ameliorate cardiac fibrosis indiabetic mice.

Example 12. Plasminogen Lowers Collagen Deposition in Heart of 17- to18-Week-Old Diabetic Mice

Eight 17- to 18-week-old male db/db mice were randomly divided into twogroups, four mice in each of the control group administered with vehiclePBS and the group administered with plasminogen. The mice were weighedand grouped on the day when the experiment began, i.e. Day 0.Plasminogen or PBS was administered from day 1 for 35 consecutive days.Mice in the group administered with plasminogen were injected withplasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein, and anequal volume of PBS was administered to mice in the control groupadministered with vehicle PBS. The mice were sacrificed afteradministration of plasminogen for 35 days. The heart tissues were fixedin 4% paraformaldehyde fixative for 24 hours. The fixed hearts wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The tissue sections was 3 μm thick. Thesections were dewaxed and rehydrated and washed with water once. Afterstained with 0.1% Sirius red in saturated picric acid for 30 min, thesections were flushed with running water for 2 min. After stained withhematoxylin for 1 min, the sections were flushed with running water,differentiated with 1% hydrochloric acid in alcohol, returned to bluewith ammonia water, flushed with running water, dried and sealed with aneutral gum. The sections were observed under an optical microscope at200×.

The results showed that the deposition of collagen fibers (indicated byarrow) in mice in the group administered with plasminogen (FIG. 12B) wasremarkably less than that in the control group administered with vehiclePBS (FIG. 12A). It indicates that plasminogen can reduce collagendeposition in the heart tissue, and suggests that plasminogen isexpected to reduce heart tissue fibrosis in relatively young (17- to18-week-old) diabetic mice by lowering collagen deposition in the hearttissue.

Example 13. Plasminogen Lowers Collagen Deposition in Heart of 26- to27-Week-Old Diabetic Mice

Nine 26- to 27-week-old male db/db mice were randomly divided into twogroups, 5 mice in the control group administered with vehicle PBS, and 4mice in the group administered with plasminogen. The mice were weighedand grouped on the day when the experiment began, i.e. Day 0.Plasminogen or PBS was administered from day 1 for 35 consecutive days.Mice in the group administered with plasminogen were injected withplasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein, and anequal volume of PBS was administered to mice in the control groupadministered with vehicle PBS. The mice were sacrificed afteradministration of plasminogen for 35 days. The heart tissues were fixedin 4% paraformaldehyde fixative for 24 hours. The fixed hearts wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The tissue sections was 3 μm thick. Thesections were dewaxed and rehydrated and washed with water once. Afterstained with 0.1% Sirius red for 60 min, the sections were flushed withrunning water. After stained with hematoxylin for 1 min, the sectionswere flushed with running water, differentiated with 1% hydrochloricacid in alcohol and returned to blue with ammonia water, flushed withrunning water, dried and sealed. The sections were observed under anoptical microscope at 200×.

The results showed that the deposition of collagen fibers (indicated byarrow) in mice in the group administered with plasminogen (FIG. 13B) wasremarkably less than that in the control group administered with vehiclePBS (FIG. 13A). It indicates that plasminogen can reduce collagendeposition in the heart tissue, and suggests that plasminogen isexpected to reduce heart tissue fibrosis in relatively old (26- to27-week-old) diabetic mice by lowering collagen deposition in the hearttissue.

Example 14. Plasminogen Lowers Renal Fibrosis in Cisplatin-Induced RenalFibrosis Model Mice

Ten healthy 8-9-week-old male C57 mice were randomly divided into twogroups, five mice in each of the control group administered with vehiclePBS and the group administered with plasminogen. After the completion ofgrouping, single intraperitoneal injection of cisplatin was performed at10 mg/Kg body weight to establish the renal fibrosis model^([30]). Afterthe model was established, mice in the group administered withplasminogen were administered with plasminogen at a dose of 1 mg/0.1mL/mouse/day via tail vein injection, and an equal volume of PBS wasadministered to mice in the control group administered with vehicle PBS.The mice were weighed and grouped on the day when the experiment began,i.e. day 0; the mice received intraperitoneal injection of cisplatin formodelling on day 1, and were administered with plasminogen or vehiclePBS 3 hours after the modelling, for an administration period of 7 days.The mice were sacrificed on Day 8. The kidneys were fixed in 4%paraformaldehyde fixative for 24 hours. The fixed kidney tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 5μm. The sections were dewaxed and rehydrated and washed with water once.The sections were repaired with citric acid for 30 minutes, and gentlyrinsed with water after cooling at room temperature for 10 minutes. Thesections were incubated with 3% hydrogen peroxide for 15 minutes, andthe tissues were circled with a PAP pen. The sections were blocked with10% goat serum (Vector laboratories, Inc., USA) for 1 hour, and afterthe time was up, the goat serum liquid was discarded. The sections wereincubated with rabbit anti-mouse IV collagen antibody (Abcam) overnightat 4° C. and washed with TBS twice for 5 minutes each time. The sectionswere incubated with a secondary antibody, goat anti-rabbit IgG (HRP)antibody (Abcam), for 1 hour at room temperature and washed with TBStwice for 5 minutes each time. The sections were developed with a DABkit (Vector laboratories, Inc., USA). After washing with water threetimes, the sections were counterstained with hematoxylin for 30 seconds,returned to blue with running water for 5 minutes, and washed with TBSonce. After dehydration with a gradient, permeabilization and sealing,the sections were observed under an optical microscope at 200×.

Cisplatin is a broad-spectrum anti-tumor drug with extensive clinicalapplication and reliable efficacy. However, it has severenephrotoxicity, mainly results in renal tubular and renal interstitialinjuries which eventually develop into renal fibrosis^([30]). Theexperimental results showed that the positive expression (indicated byarrow) of type IV collagen in the kidney in the control groupadministered with vehicle PBS (FIG. 14A) was remarkably higher than thatin the group administered with plasminogen (FIG. 14B). It indicates thatplasminogen can ameliorate renal fibrosis in cisplatin-induced renalfibrosis model mice.

Example 15. Plasminogen Ameliorates the Level of Cardiac Fibrosis inApoE Atherosclerosis Mice

Thirteen 6-week-old male ApoE mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe atherosclerosis^([31,32]). 50 μL of blood was taken from each mousethree days before administration, and the total cholesterolconcentration was detected. The mice were randomly divided into twogroups based on the detection results, 7 mice in the control groupadministered with vehicle PBS, and 6 mice in the group administered withplasminogen. The first day of administration was set as Day 1. Mice inthe group administered with plasminogen were injected with humanplasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and anequal volume of PBS was administered to mice in the control groupadministered with vehicle PBS via the tail vein. The administrationlasted for 30 days, during which mice continued to be fed with ahigh-fat and high-cholesterol diet. The mice were sacrificed on Day 31.The hearts were fixed in 4% paraformaldehyde for 24 to 48 hours. Thefixed tissues were paraffin-embedded after dehydration with alcoholgradient and permeabilization with xylene. The tissue sections was 3 μmthick. The sections were dewaxed and rehydrated and washed with wateronce. After stained with 0.1% Sirius red in saturated picric acid for 30min, the sections were flushed with running water for 2 min. Afterstained with hematoxylin for 1 min, the sections were flushed withrunning water, differentiated with 1% hydrochloric acid in alcohol,returned to blue with ammonia water, flushed with running water, driedand sealed with a neutral gum. The sections were observed under anoptical microscope at 200×.

The results showed that the collagen deposition (indicated by arrow) inthe group administered with plasminogen (FIG. 15B) was remarkably lessthan that in the control group administered with vehicle PBS (FIG. 15A),suggesting that plasminogen can prevent and lower cardiac fibrosisinduced by atherosclerosis by reducing collagen deposition in the hearttissue of ApoE atherosclerosis model mice.

Example 16. Plasminogen Lowers Cardiac Fibrosis in Hyperlipemia ModelMice

Eleven 6-week-old male C57 mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducehyperlipemia^([33,34]). 50 μL of blood was taken from each mouse threedays before administration, and the total cholesterol concentration wasdetected. The mice were randomly divided into two groups based on thedetection results, 6 mice in the control group administered with vehiclePBS, and 5 mice in the group administered with plasminogen. The firstday of administration was recorded as Day 1. Mice in the groupadministered with plasminogen were injected with human plasminogen at adose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS via the tail vein. The administration lasted for 30 days,during which mice continued to be fed with a high-fat andhigh-cholesterol diet. The mice were sacrificed on Day 31. The hearttissues were fixed in 4% paraformaldehyde for 24 to 48 hours. The fixedtissues were paraffin-embedded after dehydration with alcohol gradientand permeabilization with xylene. The tissue sections was 3 μm thick.The sections were dewaxed and rehydrated and washed with water once.After stained with 0.1% Sirius red in saturated picric acid for 30 min,the sections were flushed with running water for 2 min. After stainedwith hematoxylin for 1 min, the sections were flushed with runningwater, differentiated with 1% hydrochloric acid in alcohol, returned toblue with ammonia water, flushed with running water, dried and sealedwith a neutral gum. The sections were observed under an opticalmicroscope at 200×.

The results showed that the collagen deposition (indicated by arrow) inthe group administered with plasminogen (FIG. 16B) was remarkably lessthan that in the control group administered with vehicle PBS (FIG. 16A),suggesting that plasminogen can prevent and lower cardiac fibrosisinduced by hyperlipemia by reducing collagen deposition in the hearttissue of hyperlipemia model mice.

Example 17. Plasminogen Repairs Renal Fibrosis in Chronic Renal FailureModel

Twelve 8- to 9-week-old male mice with normal PLG activity and six malemice with impaired PLG activity were taken. The mice with normal PLGactivity were randomly divided into two groups, 6 mice in each of thegroup administered with plasminogen and the control group administeredwith vehicle PBS. Three groups of mice were fed with a 0.25% purine diet(Nantong TROPHIC) every day to establish the chronic renal failuremodel^([35]). The day of model establishment was recorded as Day 1, andadministration began at the same time. Mice in the group administeredwith plasminogen were administered with plasminogen at a dose of 1mg/0.1 mL/mouse/day, and an equal volume of PBS was administered to micein the control group administered with vehicle PBS in the same manner,both lasting for 10 consecutive days for model establishment. The micewith impaired PLG activity were not treated. The mice were sacrificed onDay 11. The kidneys were fixed in 4% paraformaldehyde for 24 hours. Thefixed kidneys were paraffin-embedded after dehydration with alcoholgradient and permeabilization with xylene. The tissue sections was 3 μmthick. The sections were dewaxed and rehydrated and washed with wateronce. After stained with 0.1% Sirius red for 60 min, the sections wereflushed with running water. After stained with hematoxylin for 1 min,the sections were flushed with running water, differentiated with 1%hydrochloric acid in alcohol and returned to blue with ammonia water,flushed with running water, dried and sealed. The sections were observedunder an optical microscope at 200×.

The results showed that the collagen deposition (indicated by arrow) inthe group administered with plasminogen (FIG. 17B) was remarkably lessthan that in the control group administered with vehicle PBS (FIG. 17A)and the group with impaired PLG activity (FIG. 17C), and the statisticaldifference between the group administered with plasminogen and the groupwith impaired PLG activity was significant (P=0.018) (FIG. 17D). Itindicates that plasminogen can significantly alleviate collagendeposition in kidney tissues of animals with chronic renal injury, thuspreventing and alleviating renal fibrosis induced by chronic renalinjury.

Example 18. Plasminogen Reduces Collagen Deposition in the PancreaticIslet of Diabetic Mice

Sixteen 24- to 25-week-old male db/db mice were randomly divided intotwo groups, 10 mice in the group administered with plasminogen, and 6mice in the control group administered with vehicle PBS. Mice in thegroup administered with plasminogen were injected with human plasminogenat a dose of 2 mg/0.2 mL/mouse/day via the tail vein, and an equalvolume of PBS was administered to mice in the control group administeredwith vehicle PBS via the tail vein. The mice were weighed and grouped onthe day when the experiment began, i.e. Day 0. Plasminogen or PBS wasadministered from day 1 for 31 consecutive days. On day 32, the micewere sacrificed, and the pancreas was taken and fixed in 4%paraformaldehyde. The fixed pancreas tissues were paraffin-embeddedafter dehydration with alcohol gradient and permeabilization withxylene. The tissue sections was 3 μm thick. The sections were dewaxedand rehydrated and washed with water once. After stained with 0.1%Sirius red for 60 min, the sections were flushed with running water.After stained with hematoxylin for 1 min, the sections were flushed withrunning water, differentiated with 1% hydrochloric acid in alcohol andreturned to blue with ammonia water, flushed with running water, driedand sealed. The sections were observed under an optical microscope at200×.

The results showed that the collagen deposition (indicated by arrow) inthe pancreatic islet of the mice in the group administered withplasminogen (FIG. 18B) was remarkably lower than that in the controlgroup administered with vehicle PBS (FIG. 18A), and the statisticaldifference was significant (FIG. 18C). It indicates that plasminogen cansignificantly alleviate collagen deposition in pancreatic tissues ofdiabetic mice, thus preventing and alleviating pancreatic injury andfibrosis.

Example 19. Plasminogen Lowers Renal Fibrosis in 3% CholesterolHyperlipemia Model Mice

Sixteen 9-week-old male C57 mice were fed with a 3% cholesterol high-fatdiet (Nantong TROPHIC) for 4 weeks to induce hyperlipemia^([30,31]).This model was designated as the 3% cholesterol hyperlipemia model. Themodel mice continued to be fed with the 3% cholesterol high-fat diet.Another five male C57 mice of the same week age were taken as the blankcontrol group, and were fed with a normal maintenance diet during theexperiment. 50 μL of blood was taken from each mouse three days beforeadministration, and the total cholesterol was detected. The model micewere randomly divided into two groups based on the total cholesterolconcentration and the body weight, i.e., the group administered withplasminogen, and the control group administered with vehicle PBS, 8 micein each group. The first day of administration was recorded as Day 1.Mice in the group administered with plasminogen were injected with humanplasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and anequal volume of PBS was administered to mice in the control groupadministered with vehicle PBS via the tail vein. The mice wereadministered for 30 days. After the mice were administered on day 30,the mice were sacrificed on Day 31. The kidney materials were taken andfixed in 4% paraformaldehyde for 24 to 48 hours. The fixed tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The sections was 3 μm thick. The sectionswere dewaxed and rehydrated and washed with water once. After stainedwith 0.1% Sirius red in saturated picric acid for 30 min, the sectionswere flushed with running water for 2 min. After stained withhematoxylin for 1 min, the sections were flushed with running water,differentiated with 1% hydrochloric acid in alcohol, returned to bluewith ammonia water, flushed with running water, dried and sealed with aneutral gum. The sections were observed under an optical microscope at200×.

The results showed that the collagen deposition in kidney (indicated byarrow) in the group administered with plasminogen (FIG. 19C) wasremarkably less than that in the control group administered with vehiclePBS (FIG. 19B), and the statistical difference was significant (FIG.19D); while in the group administered with plasminogen, fibrosis wassubstantially restored to a normal level (FIG. 19A). It indicates thatplasminogen can effectively reduce renal fibrosis in 3% cholesterolhyperlipemia model mice.

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The invention claimed is:
 1. A method for preventing or treating hepaticcollagen deposition or fibrosis caused by a hepatic tissue injury in asubject, comprising administering an effective amount of plasminogenhaving at least 75% sequence identity to the full-length sequence of SEQID NO. 2 to the subject.
 2. The method of claim 1, wherein the fibrosisis caused by an ischemic reperfusion injury, drugs, systemic immunedisease, chemicals, inflammatory response, alcoholism, cancer, or fatdeposits.
 3. The method of claim 1, wherein the plasminogen isadministered in combination with one or more other drugs.
 4. The methodof claim 3, wherein the one or more other drugs comprise: ahypolipidemic drug, an anti-platelet drug, an antihypertensive drug, avasodilator, a hypoglycemic drug, an anticoagulant drug, a thrombolyticdrug, a hepatoprotective drug, an anti-fibrosis drug, an anti-arrhythmiadrug, a cardiotonic drug, a diuretic drug, an anti-tumor drug, aradiotherapeutic or chemotherapeutic drug, an inflammatory regulatorydrug, an immunomodulatory drug, an antiviral drug, or an antibiotic. 5.The method of claim 1, wherein the plasminogen has at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% sequence identity to the full-lengthsequence of SEQ ID No. 2, and still has the plasminogen activity.
 6. Themethod of claim 1, wherein the plasminogen comprises a plasminogenactive fragment and still has the plasminogen activity.
 7. The method ofclaim 1, wherein the plasminogen is Glu-plasminogen or Lys-plasminogen.8. The method of claim 1, wherein the plasminogen is a natural orsynthetic human plasminogen.
 9. The method of claim 1, wherein theplasminogen is administered to the subject at a dosage of 1-100 mg/kg,1-50 mg/kg, or 1-10 mg/kg, daily, every other day, or weekly.
 10. Themethod of claim 9, wherein the plasminogen is administered daily. 11.The method of claim 1, wherein the plasminogen has at least 90%, 95%, or99% sequence identity to the full-length sequence of SEQ ID No.
 2. 12.The method of claim 1, wherein the plasminogen is Glu-plasminogen.
 13. Amethod for preventing or treating hepatic collagen deposition orfibrosis in a subject, comprising administering an effective amount ofplasminogen having at least 75% sequence identity to the full-lengthsequence of SEQ ID NO. 2 to the subject.
 14. The method of claim 13,wherein the hepatic collagen deposition or fibrosis is caused orcomplicated by diabetes mellitus, caused or complicated byatherosclerosis, caused or complicated by hyperlipemia, caused by drugs,or caused by chronic hepatic disease.
 15. The method of claim 14,wherein the hyperlipemia comprises one or more selected from: anelevated blood triglyceride level, an elevated total blood cholesterollevel, increased blood low-density lipoprotein, and increased blood verylow-density lipoprotein.
 16. The method of claim 14, wherein the drugsare hepatotoxic drugs.
 17. The method of claim 16, wherein the drugscomprise a chemotherapeutic drug, an antibiotic drug, a hypolipidemicdrug, an antihypertensive drug, or a hypoglycemic drug.
 18. The methodof claim 14, wherein the chronic hepatic disease is viral hepatitis. 19.A method for preventing or treating alcoholic hepatic collagendeposition, alcoholic hepatic fibrosis non-alcoholic hepatic collagendeposition or non-alcoholic hepatic fibrosis in a subject, comprisingadministering an effective amount of plasminogen having at least 75%sequence identity to the full-length sequence of SEQ ID NO. 2 to thesubject.